Methods of treating urothelial carcinoma

ABSTRACT

Methods and compositions for treating a urothelial and/or a micropapillary carcinoma, such as a micropapillary urothelial carcinoma are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/333,368, filed Jul. 16, 2014, now allowed, which claims the benefitof U.S. Provisional Application No. 61/847,532, filed Jul. 17, 2013. Thecontents of the aforesaid applications are hereby incorporated byreference in their entirety.

BACKGROUND

Cancer of the urinary bladder remains a major cause of morbidity andmortality around the world (Kaufman D S, et al. Lancet. (2009) 18374:239-49; Noon A P, et al. Nat Rev Urol. (2013) 10:67-8; Ploeg M, etal. World J Urol (2009) 27:289-93). Despite significant success oftargeted anti-cancer therapy in other common solid tumors such as breastand lung cancer, patients with loco-regionally advanced and metastaticurothelial carcinoma (UC) have limited therapy options especially whenchemoresistance develops to standard anti-cancer therapies (Gorin M A,et al. Postgrad Med. (2012) 124:28-36; Calabro F, et al. Eur Urol.(2009) 55(2):348-358).

The micropapillary variant of UC (MPUC) was first described in 1994(Amin M B, et al. Am J Surg Pathol. (1994) 18:1224-32). Encompassingapproximately 5% of all bladder cancers, MPUC is a clinically importantlesion characterized by a distinctive histology featuring smallmicropapillae created by clusters of 4 to 5 cells across, peripherallysituated nuclei and cytoplasmic vacuoles with a strong tendency todevelop intra-lymphatic permeation or simulate lymphovascularinvolvement due to the production of peri-tumoral stromal retractionartifacts (Amin M B, et al. (1994) supra); Sangoi A R, et al. Am J SurgPathol. (2010) 34:1367-76; Kamat A M, et al. Cancer (2007) 110:62-7;López J I, et al. Histopathology (1999) 34:561-2).

The diagnosis of MPUC harbors an adverse prognosis. Five year survivalafter diagnosis with metastatic urothelial carcinoma is small, pointingto the need for improved pharmacologic treatment of the disease. Thestandard cytotoxic regimen of MPUC is methotrexate, vinblastine,doxorubicin, and cisplatin (MVAC), and it warrants improvement. It iswell-accepted that the diagnosis of MPUC harbors an adverse prognosisand pathologists have strongly recommended that, even if the minority ofa urinary bladder UC features a MPUC pattern, the diagnosis of MPUC isrecommended to be made either outright or the tumor should be classifiedas UC with MPUC features (Amin M B, et al. (1994), supra; Sangoi A R, etal. (2010), supra; Kamat A M, et al. (2007), supra; López J I, et al.(1999), supra). Among the noteworthy clinicopathologic features of MPUC,is the association of metastatic disease at the time of diagnosis for atumor with either no invasion or limited invasion of the bladder wall.This finding is similar to that observed for other micropapillarycarcinomas occurring in other sites such as in the endometrium, breastand lung (del Carmen M G, et al. Gynecol Oncol. (2012) 127:651-61; ChenL, et al., Int J Surg Pathol. (2008) 16:155-63; Kamiya K, et al. ModPathol. (2008) 21:992-1001).

Given the highly aggressive nature of MPUC, the need exists fordeveloping novel therapeutic approaches for treating micropapillarycarcinomas, such as MPUC.

SUMMARY

The invention is based, at least in part, on the discovery ofalterations in the extracellular domain of a HER2 protein in aurothelial carcinoma (UC) and/or a micropapillary carcinoma, e.g.,micropapillary urothelial carcinoma (MPUC). For example, Applicants haveidentified about a 40+% prevalence of mutations of the extracellulardomain of HER2 in MPUC. In embodiments, the ERBB2 mutation frequency wassignificantly higher in UC samples having a confirmed MPUC histology(about 40% of 15 samples analyzed), compared to a lower frequency in thenon-MPUC samples (e.g., about 9% of 64 samples presenting a traditionalUC histology). In other embodiments of 64 UC cases analyzed, 3/64 had aS310F mutation and 1/64 had a ERBB2-GRB7 fusion. In certain embodiments,the alteration includes a substitution of a serine residue at position310 (S310) in HER2 by either a phenylalanine or a tyrosine residue; or asubstitution of arginine at position 157 for tryptophan. Therefore, theinvention provides, at least in part, methods for treating a urothelialand/or micropapillary carcinoma, including those of the urinary tract,urinary bladder, urothelial cells, as well as methods and reagents foridentifying, assessing or detecting an alteration as described herein,e.g., a HER2 mutation, in a urothelial and/or micropapillary urothelialcarcinoma.

Accordingly, in one aspect, the invention features, a method of treatinga subject having a urothelial cancer and/or cancer comprising ahistology of micropapillae, e.g., a urothelial and/or micropapillarycarcinoma (e.g., a micropapillary urothelial carcinoma). The methodincludes administering to the subject an effective amount of an agent(e.g., a therapeutic agent) that targets and/or inhibits HER2, e.g., aHER2 gene product (e.g., a HER2 protein), thereby treating the subject.

In one embodiment, the method further includes acquiring knowledge ofone or both of:

(i) the presence (or absence) of an alteration in HER2; or

(ii) the presence (or absence) of a micropapillary histology in thesubject, or a cancer or tumor sample from the subject.

In another embodiment, the method further includes identifying thesubject, or a cancer or tumor sample from the subject, as having one orboth of:

(i) the presence (or absence) of an alteration in HER2; or

(ii) the presence (or absence) of a micropapillary histology.

In certain embodiments, the presence of the HER2 alteration, themicropapillary histology, or both, in the subject is indicative that thesubject is likely to respond to the agent.

In yet other embodiments, the agent is administered responsive to adetermination of the presence of the HER2 alteration, the micropapillaryhistology, or both, in the subject, or the cancer or tumor sample fromthe subject.

In certain embodiments, the method further comprises acquiring knowledgethat the urothelial and/or micropapillary carcinoma (e.g., amicropapillary urothelial carcinoma) does not have a gene amplificationand/or overexpression of HER2 or a HER2 gene product.

Cancers

In certain embodiments, the cancer or carcinoma, e.g., themicropapillary carcinoma, is chosen from a cancer or carcinoma of theurinary system (e.g., kidney, bladder, ureter, urethra and urachus),urothelial cells, breast, lung, endometrium, bile duct or thyroid. Inone embodiment, carcinoma is a urothelial carcinoma (UC), e.g., ametastatic UC. In yet other embodiment, the carcinoma has amicropapillary histology and is chosen from a cancer of the urinarytract, bladder, urothelial cells or bile duct. In yet other embodiments,the carcinoma is a transitional cell carcinoma (TCC) or an urothelialcell carcinoma. In one embodiment, the carcinoma (e.g., themicropapillary carcinoma) is a micropapillary urothelial carcinoma(MPUC). It is noted that the term “micropapillary urothelial carcinoma”and its abbreviation, “MPUC” are used interchangeably herein.

In certain embodiment, the cancer has, or is identified or determined ashaving, a histology of micropapillae. In certain embodiments, themicropapillary histology or histology of micropapillae comprises smallmicropapillae created by clusters of two or more cells (typically 4 to 5cells) across, peripherally situated nuclei and cytoplasmic vacuoles.

In other embodiments, the cancer or carcinoma, e.g., the urothelialand/or micropapillary cancer or carcinoma (e.g., the micropapillaryurothelial carcinoma), comprises, or is identified or determined ashaving, an alteration in HER2, e.g., an alteration in HER2 as describedherein. In one embodiment, the micropapillary carcinoma does not have agene amplification or overexpression of HER2 or a HER2 gene product. Forexample, the cancer is not an ERBB2-amplified cancer or carcinoma (e.g.,an ERBB2-amplified urothelial carcinoma). In certain embodiments, thecancer does not have, or is identified as not having, an elevated levelof a HER2 gene product. In other embodiments, the cancer is, or isidentified as being negative for, an overexpressed HER2 gene product(e.g., the cancer is negative for HER2 amplification or overexpressionby nucleic acid or protein detection methods, e.g., PCR orimmunohistochemistry).

In other embodiments, the cancer, e.g., the micropapillary carcinoma,comprises, or is identified or determined as having, an alteration inone or more of: AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 or RAF1. In oneembodiment, the cancer has a wild-type HER2 and comprises an alterationin one or more of: AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 or RAF1. Suchcancers can be treated with modulators, e.g., inhibitors, of one or moreof: AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 or RAF1.

In certain embodiments, the alteration in HER2 results in increasedactivity of a HER2 gene product (e.g., a HER2 protein), compared to awild-type activity of HER2. For example, the alteration can result in analteration (e.g., an increase) in one or more of: kinase activity and/ordimerization of a HER2 protein. In one embodiment, the HER2 alterationis, or comprises, a mutation (e.g., a somatic mutation), e.g., asubstitution (e.g., a base substitution), a deletion or an insertion. Inone embodiment, the alteration occurs in the extracellular domain ofHER2, e.g., the alteration is found in domain II of HER2 (e.g., humanHER2). In one embodiment, the alteration is a missense mutation. In oneembodiment, the alteration is a substitution at residue 310 of HER2. Inone embodiment, the alteration is, or comprises, a substitution ofresidue S310 to phenylanine (e.g., S310F) or tyrosine (e.g., S310Y) ofHER2. In one embodiment, the alteration is, or comprises, a substitutionof residue S310 to phenylanine of HER2. In another embodiment, thealteration is, or comprises, a substitution of residue S310 to tyrosineof HER2. In yet another embodiment, the alteration is, or comprises, asubstitution of residue 157, e.g., a substitution of arginine atposition 157 for tryptophan (e.g., R157W). In yet other embodiments, thealteration is a HER2 gene amplification event. In other embodiments, thealteration is, or comprises, an alteration in the kinase domain of HER2.In other embodiments, the alteration is, or comprises, a substitution ofresidue 719, e.g., a substitution of glycine at position 719 for serine(e.g., G719S). In other embodiments, the alteration is, or comprises, asubstitution of residue 689, e.g., a substitution of valine at position689 for methionine (e.g., V689M). In other embodiments, the alterationis, or comprises, a substitution of residue 700, e.g., a substitution ofmethionine at position 700 for aspartic acid (e.g., M700D). In otherembodiments, the alteration is, or comprises, a substitution of residue826, e.g., a substitution of asparagine at position 826 for serine(e.g., N826S). In other embodiments, the alteration is, or comprises, asubstitution of residue 839, e.g., a substitution of alanine at position839 for threonine (e.g. A839T). In other embodiments, the alteration is,or comprises, a substitution of residue 861, e.g., a substitution ofleucine at position 861 for glutamine (e.g., L861Q).

Subjects

In certain embodiments, the subject has an alteration in HER2, e.g., thesubject has a urothelial and/or a micropapillary carcinoma comprising aHER2 alteration described herein. In other embodiments, the subject isidentified, or has been previously identified, as having a carcinoma(e.g., a urothelial and/or micropapillary carcinoma) comprising a HER2alteration. In one embodiment, the subject does not have a geneamplification or overexpression of HER2 or a HER2 gene product. Forexample, the subject does not have an ERBB2-amplified cancer orcarcinoma (e.g., an ERBB2-amplified urothelial carcinoma). In certainembodiments, the subject does not show an elevated level of, or isnegative for, a HER2 gene product (e.g., the cancer is negative for HER2expression by nucleic acid or protein detection methods, e.g., PCR orimmunohistochemistry).

In other embodiments, the subject is identified, or has been previouslyidentified, as having a carcinoma with a micropapillary histology, e.g.,a micropapillary histology as described herein. In yet otherembodiments, the subject is identified, or has been previouslyidentified, as having a carcinoma with an alteration in HER2 (e.g., aHER2 alternation as described herein, such as a substitution at residue310 or residue 157), and as having a carcinoma with a micropapillaryhistology. For example, the subject is identified, or has beenpreviously identified, as having a micropapillary carcinoma chosen fromthe urinary tract, bladder, urothelial cells or bile duct. In oneembodiment, the subject is identified, or has been previouslyidentified, as having an MPUC.

In one embodiment, the subject is a human. In one embodiment, thesubject has, or is at risk of having a cancer (e.g., a urothelial and/ormicropapillary carcinoma (e.g., MPUC) as described herein) at any stageof disease, e.g., a metastatic cancer. In other embodiments, the subjectis a cancer patient, e.g., a patient having a urothelial and/ormicropapillary carcinoma as described herein.

In one embodiment, the subject is undergoing or has undergone treatmentwith a different (e.g., non-HER2) therapeutic agent or therapeuticmodality. In one embodiment, the non-HER2 therapeutic agent ortherapeutic modality is a chemotherapy, immunotherapy, or a surgicalprocedure. In one embodiment, the non-HER2 therapeutic agent ortherapeutic modality comprises one or more (or all) of: methotrexate,vinblastine, doxorubicin, and/or cisplatin (MVAC).

In one embodiment, responsive to the determination of the presence ofthe HER2 alteration and/or micropapillary histology described herein,the different therapeutic agent or therapeutic modality is discontinued.In yet other embodiments, the subject has been identified as beinglikely or unlikely to respond to the different therapeutic agent ortherapeutic modality.

In certain embodiments, the subject has participated previously in aclinical trial, e.g., a clinical trial for a different (e.g., non-HER2)therapeutic agent or therapeutic modality. In other embodiments, thesubject is a cancer patient who has participated in a clinical trial,e.g., a clinical trial for a different (e.g., non-HER2) therapeuticagent or therapeutic modality.

Agents

In certain embodiments, the agent (e.g., the therapeutic agent) used inthe methods targets and/or inhibits HER2 (e.g., a HER2 gene or geneproduct as described herein). In one embodiment, the agent binds andinhibits HER2. In one embodiment, the agent is a reversible or anirreversible HER2 inhibitor. In certain embodiments, the agent is a panERBB inhibitor, or a dual or a specific HER2 inhibitor. In oneembodiment, the agent is a dual EGFR/ERBB2 inhibitor, e.g., a reversibleor an irreversible dual EGFR/ERBB2 inhibitor.

In one embodiment, the agent is an antibody molecule, e.g., an anti-HER2antibody molecule (e.g., a monoclonal or a bispecific antibody), or aconjugate thereof (e.g., an antibody to HER2 conjugated to a cytotoxicagent (e.g., mertansine DM1)). In one embodiment, the agent is a kinaseinhibitor. In one embodiment, the kinase inhibitor is chosen from: amulti-specific kinase inhibitor, a HER2/ERBB2 inhibitor, an EGFRinhibitor (e.g., a pan ERBB inhibitor), a HER3 inhibitor, and/or a smallmolecule inhibitor that is selective for HER2.

In one embodiment, the agent is chosen from: a kinase inhibitor, amulti-specific kinase inhibitor; a HER2 inhibitor; an EGFR inhibitor(e.g., a pan ERBB inhibitor); a small molecule inhibitor that isselective for HER2; an antibody molecule (e.g., a monoclonal or abispecific antibody) against HER2; an antibody to HER2 conjugated to acytotoxic agent (e.g., mertansine DM1) and/or a HER2 cellularimmunotherapy.

In one embodiment, the agent is chosen from: AV-203, AMG 888, U3-1287,APC8024, DN24-02, Neuvenge, Lapuleucel-T, MM-111, MM-121, SAR256212,MM-141, LJM716, REGN1400, MEHD7945A, RG7597, RG7116, Trastuzumab,trastuzumab emtansine (T-DM1), pertuzumab, afatinib, TAK-285, Neratinib,Dacomitinib, BMS-690514, BMS-599626, Pelitinib, CP-724714, Lapatinib,TAK-165, ARRY-380, and/or AZD8931. In one embodiment, the agent is anirreversible HER2/EGFR tyrosine kinase inhibitor, e.g., Neratinib.

In other embodiments, the agent is chosen from a nucleic acid molecule(e.g., an antisense molecule, a ribozyme, a double stranded RNA, or atriple helix molecule) that hybridizes to and/or inhibits a HER2 nucleicacid, e.g., a HER2 nucleic acid encoding the alteration, or atranscription regulatory region that blocks or reduces mRNA expressionof the alteration.

Compositions, e.g., pharmaceutical compositions, comprising one or moreof the agents, e.g., the therapeutic agents described herein, for use,e.g., in treating a urothelial and/or micropapillary carcinoma (e.g.,MPUC) as described herein are also disclosed.

Additionally, kits comprising the agents, e.g., the therapeutic agents(and compositions thereof), with instructions for use in treating aurothelial and/or micropapillary carcinoma (e.g., MPUC) and/ordetermining the presence of an alteration and/or a histology describedherein are also provided.

In another aspect, the invention features a kit comprising one or moredetection reagents (e.g., probes, primers, antibodies), capable, e.g.,of specific detection of a nucleic acid or protein comprising analteration described herein.

The invention also provides methods of: identifying, assessing ordetecting an alteration described herein, e.g., a HER2 mutation, in aurothelial and/or micropapillary carcinoma (e.g., MPUC). Included areisolated nucleic acid molecules comprising the alterations, nucleic acidconstructs, host cells containing the nucleic acid molecules; purifiedpolypeptides comprising the alteration described herein and bindingagents; detection reagents (e.g., probes, primers, antibodies, kits,capable, e.g., of specific detection of a nucleic acid or proteincomprising an alteration described herein); screening assays foridentifying molecules that interact with, e.g., inhibit the alterations,e.g., novel kinase inhibitors or binders of HER2. In one embodiment, thedetection of the alteration comprises sequencing, e.g., nucleic acidsequencing or amino acid sequencing.

Alternatively, or in combination with the methods described herein, theinvention features a method of determining the presence of an alterationand/or micropapillary histology described herein in a cancer, e.g., aurothelial and/or micropapillary carcinoma (e.g., micropapillaryurothelial carcinoma (MPUC)). The method includes: acquiring knowledge(e.g., directly acquiring knowledge) that the alteration describedherein is present in a subject, e.g., a sample (e.g., a cancer or tumorsample) from the subject. In one embodiment, the acquiring stepcomprises a determination of the presence of the alteration in a nucleicacid molecule from the subject, e.g., by performing a sequencing step.In other embodiments, the acquiring step comprises a determination ofthe presence of a polypeptide or a protein comprising the alterationdescribed herein in the sample from the subject. Alternatively or incombination, the method further includes determining the histology ofthe sample, e.g., determining if the sample has a micropapillaryhistology.

Additional aspects or embodiments of the invention include one or moreof the following.

In one embodiment, the subject, or the sample, comprises one or morecells or tissue from a urothelial and/or micropapillary carcinoma chosenfrom the urinary tract, bladder, urothelial cells or bile duct. In oneembodiment, the subject or sample comprises one or more cells or tissuefrom a urothelial or a micropapillary urothelial carcinoma.

In one embodiment the method further comprises administering an agent,e.g., a therapeutic agent that targets and/or inhibits HER2, e.g., anagent as described herein, to the subject responsive to thedetermination of the presence of the alteration and/or themicropapillary histology in the sample from the subject.

In one embodiment, the mutation is detected in a nucleic acid moleculeor a polypeptide. The method includes detecting whether a mutatednucleic acid molecule or polypeptide is present in a cell (e.g., acirculating cell), a tissue (e.g., a tumor), or a sample, e.g., a tumorsample, from a subject. In one embodiment, the sample is a nucleic acidsample. In one embodiment, the nucleic acid sample comprises DNA, e.g.,genomic DNA or cDNA, or RNA, e.g., mRNA. In other embodiments, thesample is a protein sample.

In embodiments, the method further includes determining the histology ofthe tissue or the sample, e.g., determining if the sample has amicropapillary histology. In one embodiment, the sample is, or has been,classified as having a micropapillary histology.

In one embodiment, the sample or tissue is, or has been, classified asnon-malignant or malignant using other diagnostic techniques, e.g.,immunohistochemistry. For example, the sample or tissue does not show anelevated level of, or is negative for, a HER2 gene product (e.g., thecancer is negative for HER2 expression by nucleic acid or proteindetection methods, e.g., PCR or immunohistochemistry).

In one embodiment, the sample is acquired from a subject (e.g., asubject having or at risk of having a cancer, e.g., a patient), oralternatively, the method further includes acquiring a sample from thesubject. The sample can be chosen from one or more of: tissue, e.g.,cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma,buccal scrape, sputum, saliva, cerebrospinal fluid, urine, stool,circulating tumor cells, circulating nucleic acids, or bone marrow. Incertain embodiments, the sample is a tissue (e.g., a tumor biopsy), acirculating tumor cell or nucleic acid.

In embodiments, the tumor is from a cancer described herein, e.g., ischosen from a urothelial and/or micropapillary carcinoma, e.g., a MPUC.

In one embodiment, the subject is at risk of having, or has a urothelialand/or micropapillary carcinoma, e.g., a MPUC.

In other embodiments, the mutation is detected in a nucleic acidmolecule by a method chosen from one or more of: nucleic acidhybridization assay, amplification-based assays (e.g., polymerase chainreaction (PCR)), PCR-RFLP assay, real-time PCR, sequencing, screeninganalysis, SSP, HPLC or mass-spectrometric genotyping.

In one embodiment, the method includes: contacting a nucleic acidsample, e.g., a genomic DNA sample (e.g., a chromosomal sample or afractionated, enriched or otherwise pre-treated sample) or a geneproduct (mRNA, cDNA), obtained from the subject, with a nucleic acidfragment (e.g., a probe or primer as described herein (e.g., anexon-specific probe or primer) under conditions suitable forhybridization, and determining the presence or absence of the mutatednucleic acid molecule. The method can, optionally, include enriching asample for the gene or gene product.

Alternatively, or in combination with the methods described herein, theinvention features a method for determining the presence of a mutatednucleic acid molecule. The method includes: acquiring a sequence for aposition in a nucleic acid molecule, e.g., by sequencing at least onenucleotide of the nucleic acid molecule (e.g., sequencing at least onenucleotide in the nucleic acid molecule that comprises the mutation),thereby determining that the mutation is present in the nucleic acidmolecule. Optionally, the sequence acquired is compared to a referencesequence, or a wild type reference sequence. In one embodiment, thenucleic acid molecule is from a cell (e.g., a circulating cell), atissue (e.g., a urothelial and/or micropapillary carcinoma, e.g., aMPUC), or any sample from a subject (e.g., blood or plasma sample). Inother embodiments, the nucleic acid molecule from a tumor sample (e.g.,a tumor or cancer sample) is sequenced. In one embodiment, the sequenceis determined by a next generation sequencing method. The method furthercan further include acquiring, e.g., directly or indirectly acquiring, asample, e.g., a urothelial and/or micropapillary carcinoma, e.g., aMPUC.

In another aspect, the invention features a method of analyzing a tumoror a circulating tumor cell. The method includes acquiring a nucleicacid sample from the tumor or the circulating cell; and sequencing,e.g., by a next generation sequencing method, a nucleic acid molecule,e.g., a nucleic acid molecule that includes an alteration as describedherein.

In yet other embodiment, a polypeptide comprising an alterationdescribed herein is detected. The method includes: contacting a proteinsample with a reagent which specifically binds to a polypeptidecomprising an alteration described herein; and detecting the formationof a complex of the polypeptide and the reagent. In one embodiment, thereagent is labeled with a detectable group to facilitate detection ofthe bound and unbound reagent. In one embodiment, the reagent is anantibody molecule, e.g., is selected from the group consisting of anantibody, and antibody derivative, and an antibody fragment.

In yet another embodiment, the level (e.g., expression level) oractivity the polypeptide comprising an alteration described herein isevaluated. For example, the level (e.g., expression level) or activityof the polypeptide (e.g., mRNA or polypeptide) is detected and(optionally) compared to a pre-determined value, e.g., a reference value(e.g., a control sample).

In yet another embodiment, the alteration is detected prior toinitiating, during, or after, a treatment in a subject having analteration described herein.

In one embodiment, the alteration is detected at the time of diagnosiswith a cancer. In other embodiment, the alteration is detected at apre-determined interval, e.g., a first point in time and at least at asubsequent point in time.

In certain embodiments, responsive to a determination of the presence ofthe alteration, any of the methods described herein further include oneor more of:

(1) stratifying a patient population (e.g., assigning a subject, e.g., apatient, to a group or class);

(2) identifying or selecting the subject as being likely or unlikely torespond to a treatment, e.g., a HER2 inhibitor treatment as describedherein;

(3) selecting a treatment option, e.g., administering or notadministering a preselected therapeutic agent, e.g., a HER2 inhibitor asdescribed herein; or

(4) prognosticating the time course of the disease in the subject (e.g.,evaluating the likelihood of increased or decreased patient survival).

In certain embodiments, responsive to the determination of the presenceof a mutation, the subject is classified as a candidate to receivetreatment with a therapy disclosed herein. In one embodiment, responsiveto the determination of the presence of a mutation, the subject, e.g., apatient, can further be assigned to a particular class if a mutation isidentified in a sample of the patient. For example, a patient identifiedas having a mutation can be classified as a candidate to receivetreatment with a therapy disclosed herein. In one embodiment, thesubject, e.g., a patient, is assigned to a second class if the mutationis not present. For example, a patient who has a tumor that does notcontain a mutation, may be determined as not being a candidate toreceive a therapy disclosed herein.

In another embodiment, responsive to the determination of the presenceof the alteration, the subject is identified as likely to respond to atreatment that comprises a therapy disclosed herein.

In yet another embodiment, responsive to the determination of thepresence of the alteration, the method includes administering an agent,e.g., a therapeutic agent as described herein, e.g., a HER2 inhibitor,to the subject.

Method of Evaluating a Tumor or a Subject

In another aspect, the invention features a method of evaluating asubject (e.g., a patient), e.g., for risk of having or developing acancer, e.g., a urothelial and/or micropapillary carcinoma, e.g., MPUC.The method includes: acquiring information or knowledge of the presenceof a mutation as described herein in a subject (e.g., acquiring genotypeinformation of the subject that identifies a mutation as being presentin the subject); acquiring a sequence for a nucleic acid moleculeidentified herein (e.g., a nucleic acid molecule that includes amutation sequence); or detecting the presence of a nucleic acid orpolypeptide in the subject), wherein the presence of the mutation ispositively correlated with increased risk for, or having, a cancerassociated with such a mutation.

The method can further include acquiring, e.g., directly or indirectly,a sample from a patient and evaluating the sample for the present of analteration and/or a micropapillary histology as described herein.

The method can further include the step(s) of identifying (e.g.,evaluating, diagnosing, screening, and/or selecting) the subject asbeing positively correlated with increased risk for, or having, a cancerassociated with the alteration.

In another embodiment, a subject identified as having the alterationand/or micropapillary histology is identified or selected as likely orunlikely to respond to a treatment, e.g., a therapy disclosed herein.The method can further include treating the subject with a therapydisclosed herein.

In a related aspect, a method of evaluating a patient or a patientpopulation is provided. The method includes: identifying, selecting, orobtaining information or knowledge that the patient or patientpopulation has participated in a clinical trial; acquiring informationor knowledge of the presence of an alteration (e.g., an alteration asdescribed herein) in the patient or patient population (e.g., acquiringgenotype information of the subject that identifies an alteration asbeing present in the subject); acquiring a sequence for a nucleic acidmolecule identified herein (e.g., a nucleic acid molecule that includesan alteration sequence); or detecting the presence of a mutated nucleicacid or polypeptide in the subject), wherein the presence of thealteration, alone or in combination with a micropapillary histology,identifies the patient or patient population as being likely to respondto an agent as described herein (e.g., a HER2 inhibitor).

In some embodiments, the method further includes treating the subjectwith an agent as described herein (e.g., a HER2 inhibitor).

Reporting

Methods described herein can include providing a report, such as, inelectronic, web-based, or paper form, to the patient or to anotherperson or entity, e.g., a caregiver, e.g., a physician, e.g., anoncologist, a hospital, clinic, third-party payor, insurance company orgovernment office. The report can include output from the method, e.g.,the identification of nucleotide values, the indication of presence orabsence of an alteration and/or micropapillary histology as describedherein, or wildtype sequence. In one embodiment, a report is generated,such as in paper or electronic form, which identifies the presence orabsence of an alteration described herein, and optionally includes anidentifier for the patient from which the sequence was obtained.

The report can also include information on the role of a mutation asdescribed herein, or wildtype sequence, in disease. Such information caninclude information on prognosis, resistance, or potential or suggestedtherapeutic options, e.g., an agent as described herein (e.g., a HER2inhibitor). The report can include information on the likelyeffectiveness of a therapeutic option, the acceptability of atherapeutic option, or the advisability of applying the therapeuticoption to a patient, e.g., a patient having a sequence, alteration ormutation identified in the test, and in embodiments, identified in thereport. For example, the report can include information, or arecommendation on, the administration of a drug, e.g., theadministration at a preselected dosage or in a preselected treatmentregimen, e.g., in combination with other drugs, to the patient. In oneembodiment, not all mutations identified in the method are identified inthe report. For example, the report can be limited to mutations in geneshaving a preselected level of correlation with the occurrence,prognosis, stage, or susceptibility of the cancer to treatment, e.g.,with a preselected therapeutic option. The report can be delivered,e.g., to an entity described herein, within 7, 14, or 21 days fromreceipt of the sample by the entity practicing the method.

In another aspect, the invention features a method for generating areport, e.g., a personalized cancer treatment report, by obtaining asample, e.g., a tumor sample, from a subject, detecting a mutation asdescribed herein in the sample, and selecting a treatment based on themutation identified. In one embodiment, a report is generated thatannotates the selected treatment, or that lists, e.g., in order ofpreference, two or more treatment options based on the mutationidentified. In another embodiment, the subject, e.g., a patient, isfurther administered the selected method of treatment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, and theexample are illustrative only and not intended to be limiting.

The details of one or more embodiments featured in the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages featured in the invention will beapparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a tile plot of genomic alterations in 15 cases ofmicropapillary urothelial carcinoma.

FIGS. 2A-2D depicts a Table summarizing the clinical features andgenomic alterations identified in 15 MUPC cases. The data for each caseis encompassed in FIG. 2A-2D. For each study case, the clinical featuresof: gender (female (F) or male (M), age at the time the sample wasobtained, specimen sequenced, tumor type, tumor grade, tumor stagecoverage depth, genomic alterations, actionable alterations, and thegenomic alterations in AKT1, AKT2, and ARIDIA are summarized for eachstudy case in FIG. 2A. For each study case, the genomic alterations inAURKA, BAP1, CCND1, CCND3, CCNE1, EGFR, EPHA3, ERBB2, FBXW, HRAS, andIDHA are summarized in FIG. 2B. For each study case, the genomicalterations in IRS2, JAK2, KRAS, MCL1, MDM2, MLL2, MSH2, MYCL1, NF2,PTCH1, PIK3CA, and PIK3R1 are summarized in FIG. 2C. For each studycase, the genomic alterations in PTEN, RAF1, and RB1 are summarized inFIG. 2D.

For example, for Study Case 1, the clinical features of: gender (F), ageat the time the samples was obtained (71), specimen sequenced(metastasis) tumor type (MPUC), tumor grade (HG), tumor stage (IV),coverage depth (1525), genomic alterations (3), actionable alterations(2), and the genomic alterations in AKT1 (none), AKT2 (none), and ARIDIA(none) are summarized for each study case in FIG. 2A. For each studycase, the genomic alterations in AURKA (none), BAP1 (none), CCND1(none), CCND3 (none), CCNE1 (none), EGFR (none), EPHA3 (none), ERBB2(S310F), FBXW (G423V), HRAS (none), and IDHA (none) are summarized inFIG. 2B. For each study case, the genomic alterations in IRS2 (none),JAK2 (none), KRAS (none), MCL1 (none), MDM2 (none), MLL2 (none), MSH2(none), MYCL1 (none), NF2 (none), PTCH1 (none), PIK3CA (none), andPIK3R1 (none) are summarized in FIG. 2C. For each study case, thegenomic alterations in PTEN (none), RAF1 (none), and RB1 (none) aresummarized in FIG. 2D.

FIG. 3 depicts the relative incidence of erbb2 mutations in lung cancer,breast cancer, urinary bladder cancer (all urothelial carcinomas) in thecosmic database and micropapillary urothelial carcinoma in the currentstudy.

FIGS. 4A-4Z depicts a Table summarizing the genomic alterations in 64cases of relapsed/metastatic non-micropapillary urothelial carcinoma ofthe bladder. The data for each case is encompassed in two consecutivefigures. For example, the data for cases N1-N4 is encompassed in FIG.4A-4B; the data describing the histology, sample type, known shortvariants, and likely short variants for cases N1-N4 is encompassed inFIG. 4A; and the data describing known CNAs, known rearrangements, andlikely rearrangements for cases N1-N4 is encompassed in FIG. 4B. Forfurther example, for study case N1 the data describing the histology(non-MPUC), sample type (TURBT), known short variants(FGFR1_c.422C>G_p.T141R(0.52,660)), and likely short variants(MLL2:NM_003482:c.15597_15612delGGCAGTGGCACTATGA_p.H5200fs*38(0.29,664), TP53:NM_000546:c.559+1C>A_p.splice(0.41,530)) is encompassed in FIG. 4A; and the datadescribing known CNAs (CCND1_amplification(9,exons 5 of 5),CDKN2A_loss(0,exons 5 of 5), CDKN2B_loss(0,exons 5 of 5),ERBB2_amplification(20,exons 27 of 27), FGF19_amplification(9,exons 3 of3), FGF3_amplification(9,exons 3 of 3), FGF4_amplification(9,exons 3 of3), RAF1_amplification(16,exons 16 of 16)), known rearrangements (none),and likely rearrangements (none) is encompassed in FIG. 4B.

FIG. 5 depicts the Histology and List of Genomic Alterations in 6 casesof Micropapillary Urothelial Carcinoma Featuring Mutations in the ERBB2Gene.

FIG. 6 depicts a tile plot of genomic alterations in cases ofmicropapillary urothelial carcinoma.

DETAILED DESCRIPTION

Described herein is the identification of a mutation at a Serine atposition 310 to phenylalanine (S310F) in the extracellular domain ofHER2 in a human with urothelial carcinoma (UC). Additionally describedherein is a genomic analysis of a series of patients with micropapillaryurothelial carcinoma (MPUC) and non-micropapillary urothelial bladdercarcinomas (non-MPUC) to characterize the genomic landscape of MPUC. Ina retrospective series of MPUC, Applicants have identified about 40+%prevalence of mutations of the extracellular domain of HER2. Inembodiments, the ERBB2 mutation frequency was significantly higher in UCsamples having a confirmed MPUC histology (about 40% of 15 samplesanalyzed), compared to a lower frequency in the non-MPUC samples (e.g.,about 9% of 64 samples presenting a traditional UC histology). Inparticular, the Serine at position 310 is mutated to phenylalanine(S310F) or tyrosine (S310Y), and other functional mutations of HER2 arealso observed. ERBB2 S310F is an activating HER2 mutation, which issensitive to irreversible dual Egfr/Erbb2 inhibitors. ERBB2 mutationshave not been previously reported in urothelial carcinoma (COSMIC,PubMed, August 2012), yet may suggest sensitivity to Her2-targeted drugtherapies. These results further suggest a significant correlation ofgenotype to histologic phenotype and biologic behavior in an aggressivegenitourinary neoplasm, a well delineated paradigm of HER2 mutation asan oncogenic driving genomic alteration.

Accordingly, disclosed herein are methods for treating for a urothelialand/or micropapillary carcinoma, including those of the urinary tract,bladder, urothelial cells, such as micropapillary urothelial carcinoma,using an agent (e.g., a therapeutic agent) that targets and/or inhibitsHER2 (e.g., a HER2 gene product, e.g., a HER2 protein), as well asmethods and reagents for identifying, assessing and/or detecting analteration as described herein, e.g., a HER2 mutation, in amicropapillary carcinoma (e.g., micropapillary urothelial carcinoma).

Micropapillary morphology or histology occurs in neoplasms arising indifferent organ systems and displays aggressive biologic behaviorregardless of its site of origin. Carcinomas with this morphologyinclude those affecting organs and tissues, such as the urinary tract,bladder, urothelial cells, bile duct, thyroid, endometrium, breast andlung. An evaluation of a micropapillary morphology or histology can becarried out using methods known in the art as described in, for example,De Oliveira, R. et al. (2009) American Journal of Clinical Pathology,131, 694-700 (for lung adenocarcinoma); and Singh K. et al. DiagnosticPathology 2011, 6:13 (for endometrium)).

Micropapillary urothelial carcinoma of the urinary bladder is a rare,but highly aggressive form of bladder cancer associated with distantmetastases and shortened patient survival. The micropapillary morphologyor histology in the context of MPUC can be characterized by adistinctive histology featuring small micropapillae created by clustersof 4 to 5 cells across, peripherally situated nuclei and cytoplasmicvacuoles with a strong tendency to develop intra-lymphatic permeation orsimulate lymphovascular involvement due to the production ofperi-tumoral stromal retraction artifacts (described in, for example,(Amin M B, et al. (1994) Am J Surg Pathol. 18:1224-32); Sangoi A R, etal. (2010) Am J Surg Pathol. 34:1367-76; Kamat A M, et al. (2007) Cancer110:62-7; López J I, et al. (1999) Histopathology 34:561-2).

“Human Epidermal Growth Factor Receptor 2” or “HER2” (also known as Neu,ErbB-2, CD340 (cluster of differentiation 340) or p185) refers to a HER2molecule (e.g., a nucleic acid or protein). The HER2 protein refers to aprotein, typically human HER2 that is encoded by the ERBB2 gene. HER2 isa member of the epidermal growth factor receptor (EGFR/ErbB) family. TheERBB family of receptor tyrosine kinases contains four known members:Epidermal Growth Factor receptor (EGFR, ERBB1, HER1); ERRB2 (HER2),ERBB3 (HER3), and ERBB4 (HER4). HER2 protein is about 1255 amino acidsin length. The HER2 amino and nucleotide sequences are known in the art(see e.g., Coussens L. et al. (1985) Science 230 (4730): 1132-9, and arereproduced herein below).

Amplification or over-expression of the ERBB2 gene has been shown toplay an important role in the pathogenesis and progression of certainaggressive types of breast cancer. In recent years, it has evolved tobecome an important biomarker and target of therapy for the disease.Additional HER2 somatic mutations have been shown to be activatingmutations that are likely to achieve tumorigenesis (see e.g., Bose etal. Cancer Discov. 3(2):224-37). Exemplary somatic mutations thatactivate the ERBB2 signaling can be divided into at least three types ofsmall insertions and missense mutations in the kinase domain; missensemutations in the extracellular domain; and large deletions of theextracellular domain that yield the truncated form of ERBB2 (p95HER2)(Herter-Sprie G S, et al. (2013) Front Oncol. 3:86). The 5310 mutationis considered to be an activating mutation, sensitive to irreversibledual EGFR/ERBB2 inhibitors (Lee J C, et al. (2006) PLoS Med. 3:e485;Greulich H. et al. (2010) Genes Cancer. 1:1200-10; Greulich H, et al.(2012) Proc Natl Acad Sci USA. 109:14476-81).

Certain terms are defined below and throughout the specification.

As used herein, the articles “a” and “an” refer to one or to more thanone (e.g., to at least one) of the grammatical object of the article.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a physical entity, or a value, e.g., a numericalvalue, by “directly acquiring” or “indirectly acquiring” the physicalentity or value. “Directly acquiring” means performing a process (e.g.,performing a synthetic or analytical method) to obtain the physicalentity or value. “Indirectly acquiring” refers to receiving the physicalentity or value from another party or source (e.g., a third partylaboratory that directly acquired the physical entity or value).Directly acquiring a physical entity includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial. Exemplary changes include making a physical entity from two ormore starting materials, shearing or fragmenting a substance, separatingor purifying a substance, combining two or more separate entities into amixture, performing a chemical reaction that includes breaking orforming a covalent or non-covalent bond. Directly acquiring a valueincludes performing a process that includes a physical change in asample or another substance, e.g., performing an analytical processwhich includes a physical change in a substance, e.g., a sample,analyte, or reagent (sometimes referred to herein as “physicalanalysis”), performing an analytical method, e.g., a method whichincludes one or more of the following: separating or purifying asubstance, e.g., an analyte, or a fragment or other derivative thereof,from another substance; combining an analyte, or fragment or otherderivative thereof, with another substance, e.g., a buffer, solvent, orreactant; or changing the structure of an analyte, or a fragment orother derivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the analyte; orby changing the structure of a reagent, or a fragment or otherderivative thereof, e.g., by breaking or forming a covalent ornon-covalent bond, between a first and a second atom of the reagent.

“Acquiring a sequence” as the term is used herein, refers to obtainingpossession of a nucleotide sequence or amino acid sequence, by “directlyacquiring” or “indirectly acquiring” the sequence. “Directly acquiring asequence” means performing a process (e.g., performing a synthetic oranalytical method) to obtain the sequence, such as performing asequencing method (e.g., a Next Generation Sequencing (NGS) method).“Indirectly acquiring a sequence” refers to receiving information orknowledge of, or receiving, the sequence from another party or source(e.g., a third party laboratory that directly acquired the sequence).The sequence acquired need not be a full sequence, e.g., sequencing ofat least one nucleotide, or obtaining information or knowledge thatidentifies a mutation disclosed herein as being present in a subjectconstitutes acquiring a sequence.

Directly acquiring a sequence includes performing a process thatincludes a physical change in a physical substance, e.g., a startingmaterial, such as a tissue sample, e.g., a biopsy, or an isolatednucleic acid (e.g., DNA or RNA) sample. Exemplary changes include makinga physical entity from two or more starting materials, shearing orfragmenting a substance, such as a genomic DNA fragment; separating orpurifying a substance (e.g., isolating a nucleic acid sample from atissue); combining two or more separate entities into a mixture,performing a chemical reaction that includes breaking or forming acovalent or non-covalent bond. Directly acquiring a value includesperforming a process that includes a physical change in a sample oranother substance as described above.

“Acquiring a sample” as the term is used herein, refers to obtainingpossession of a sample, e.g., a tissue sample or nucleic acid sample, by“directly acquiring” or “indirectly acquiring” the sample. “Directlyacquiring a sample” means performing a process (e.g., performing aphysical method such as a surgery or extraction) to obtain the sample.“Indirectly acquiring a sample” refers to receiving the sample fromanother party or source (e.g., a third party laboratory that directlyacquired the sample). Directly acquiring a sample includes performing aprocess that includes a physical change in a physical substance, e.g., astarting material, such as a tissue, e.g., a tissue in a human patientor a tissue that has was previously isolated from a patient. Exemplarychanges include making a physical entity from a starting material,dissecting or scraping a tissue; separating or purifying a substance(e.g., a sample tissue or a nucleic acid sample); combining two or moreseparate entities into a mixture; performing a chemical reaction thatincludes breaking or forming a covalent or non-covalent bond. Directlyacquiring a sample includes performing a process that includes aphysical change in a sample or another substance, e.g., as describedabove.

An “alteration” as used herein, of a gene or gene product (e.g., a HER2gene or gene product) refers to the presence of a mutation or mutationswithin the gene or gene product, e.g., a mutation, which affects amountor activity of the gene or gene product, as compared to the normal orwild-type gene. The alteration can be in amount, structure, and/oractivity in a cancer tissue or cancer cell, as compared to its amount,structure, and/or activity, in a normal or healthy tissue or cell (e.g.,a control), and is associated with a disease state, such as cancer. Forexample, a gene or gene product which is associated with cancer, orpredictive of responsiveness to anti-cancer therapeutics, can have analtered nucleotide sequence (e.g., a mutation), amino acid sequence,chromosomal translocation, intra-chromosomal inversion, copy number,expression level, protein level, protein activity, or methylationstatus, in a cancer tissue or cancer cell, as compared to a normal,healthy tissue or cell. Exemplary mutations include, but are not limitedto, point mutations (e.g., silent, missense, or nonsense), deletions,insertions, inversions, linking mutations, duplications, translocations,inter- and intra-chromosomal rearrangements. Mutations can be present inthe coding or non-coding region of the gene. In certain embodiments, thealterations are associated (or not associated) with a phenotype, e.g., acancerous phenotype (e.g., one or more of cancer risk, cancerprogression, cancer treatment or resistance to cancer treatment).

“Binding entity” means any molecule to which molecular tags can bedirectly or indirectly attached that is capable of specifically bindingto an analyte. The binding entity can be an affinity tag on a nucleicacid sequence. In certain embodiments, the binding entity allows forseparation of the nucleic acid from a mixture, such as an avidinmolecule, or an antibody that binds to the hapten or an antigen-bindingfragment thereof. Exemplary binding entities include, but are notlimited to, a biotin molecule, a hapten, an antibody, an antibodybinding fragment, a peptide, and a protein.

“Complementary” refers to sequence complementarity between regions oftwo nucleic acid strands or between two regions of the same nucleic acidstrand. It is known that an adenine residue of a first nucleic acidregion is capable of forming specific hydrogen bonds (“base pairing”)with a residue of a second nucleic acid region which is antiparallel tothe first region if the residue is thymine or uracil. Similarly, it isknown that a cytosine residue of a first nucleic acid strand is capableof base pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.In certain embodiments, the first region comprises a first portion andthe second region comprises a second portion, whereby, when the firstand second portions are arranged in an antiparallel fashion, at leastabout 50%, at least about 75%, at least about 90%, or at least about 95%of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. In otherembodiments, all nucleotide residues of the first portion are capable ofbase pairing with nucleotide residues in the second portion.

The term “cancer” or “tumor” is used interchangeably herein. These termsrefer to the presence of cells possessing characteristics typical ofcancer-causing cells, such as uncontrolled proliferation, immortality,metastatic potential, rapid growth and proliferation rate, and certaincharacteristic morphological features.

The term “neoplasm” or “neoplastic” cell refers to an abnormalproliferative stage, e.g., a hyperproliferative stage, in a cell ortissue that can include a benign, pre-malignant, malignant (cancer) ormetastatic stage.

Cancer is “inhibited” if at least one symptom of the cancer isalleviated, terminated, slowed, or prevented. As used herein, cancer isalso “inhibited” if recurrence or metastasis of the cancer is reduced,slowed, delayed, or prevented.

“Chemotherapeutic agent” means a chemical substance, such as a cytotoxicor cytostatic agent that is used to treat a condition, particularlycancer.

As used herein, “cancer therapy” and “cancer treatment” are synonymousterms.

As used herein, “chemotherapy” and “chemotherapeutic” and“chemotherapeutic agent” are synonymous terms.

The terms “homology” or “identity,” as used interchangeably herein,refer to sequence similarity between two polynucleotide sequences orbetween two polypeptide sequences, with identity being a more strictcomparison. The phrases “percent identity or homology” and “% identityor homology” refer to the percentage of sequence similarity found in acomparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value there between.Identity or similarity can be determined by comparing a position in eachsequence that can be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical or matching nucleotides at positions sharedby the polynucleotide sequences. A degree of identity of polypeptidesequences is a function of the number of identical amino acids atpositions shared by the polypeptide sequences. A degree of homology orsimilarity of polypeptide sequences is a function of the number of aminoacids at positions shared by the polypeptide sequences. The term“substantially identical,” as used herein, refers to an identity orhomology of at least 75%, at least 80%, at least 85%, at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

“Likely to” or “increased likelihood,” as used herein, refers to anincreased probability that an item, object, thing or person will occur.Thus, in one example, a subject that is likely to respond to treatmentwith a kinase inhibitor, alone or in combination, has an increasedprobability of responding to treatment with the inhibitor alone or incombination, relative to a reference subject or group of subjects.

“Unlikely to” refers to a decreased probability that an event, item,object, thing or person will occur with respect to a reference. Thus, asubject that is unlikely to respond to treatment with a kinaseinhibitor, alone or in combination, has a decreased probability ofresponding to treatment with a kinase inhibitor, alone or incombination, relative to a reference subject or group of subjects.

“Sequencing” a nucleic acid molecule requires determining the identityof at least 1 nucleotide in the molecule. In embodiments, the identityof less than all of the nucleotides in a molecule is determined. Inother embodiments, the identity of a majority or all of the nucleotidesin the molecule is determined.

“Next-generation sequencing or NGS or NG sequencing” as used herein,refers to any sequencing method that determines the nucleotide sequenceof either individual nucleic acid molecules (e.g., in single moleculesequencing) or clonally expanded proxies for individual nucleic acidmolecules in a highly parallel fashion (e.g., greater than 10⁵ moleculesare sequenced simultaneously). In one embodiment, the relative abundanceof the nucleic acid species in the library can be estimated by countingthe relative number of occurrences of their cognate sequences in thedata generated by the sequencing experiment. Next generation sequencingmethods are known in the art, and are described, e.g., in Metzker, M.(2010) Nature Biotechnology Reviews 11:31-46, incorporated herein byreference. Next generation sequencing can detect a variant present inless than 5% of the nucleic acids in a sample.

“Sample,” “tissue sample,” “patient sample,” “patient cell or tissuesample” or “specimen” each refers to a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample can be solid tissue as from a fresh, frozen and/or preservedorgan, tissue sample, biopsy, or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid or interstitial fluid; or cells from any time ingestation or development of the subject. The tissue sample can containcompounds that are not naturally intermixed with the tissue in naturesuch as preservatives, anticoagulants, buffers, fixatives, nutrients,antibiotics or the like. In one embodiment, the sample is preserved as afrozen sample or as formaldehyde- or paraformaldehyde-fixedparaffin-embedded (FFPE) tissue preparation. For example, the sample canbe embedded in a matrix, e.g., an FFPE block or a frozen sample.

A “tumor nucleic acid sample” as used herein, refers to nucleic acidmolecules from a tumor or cancer sample. Typically, it is DNA, e.g.,genomic DNA, or cDNA derived from RNA, from a tumor or cancer sample. Incertain embodiments, the tumor nucleic acid sample is purified orisolated (e.g., it is removed from its natural state).

A “control” or “reference” “nucleic acid sample” as used herein, refersto nucleic acid molecules from a control or reference sample. Typically,it is DNA, e.g., genomic DNA, or cDNA derived from RNA, not containingthe alteration or variation in the gene or gene product, e.g., notcontaining a mutation. In certain embodiments, the reference or controlnucleic acid sample is a wild type or a non-mutated sequence. In certainembodiments, the reference nucleic acid sample is purified or isolated(e.g., it is removed from its natural state). In other embodiments, thereference nucleic acid sample is from a non-tumor sample, e.g., a bloodcontrol, a normal adjacent tumor (NAT), or any other non-canceroussample from the same or a different subject.

“Adjacent to the interrogation position,” as used herein, means that asite sufficiently close such that a detection reagent complementary withthe site can be used to distinguish between a mutation, e.g., analteration described herein, and a reference sequence, e.g., anon-mutant or wild-type sequence, in a target nucleic acid. Directlyadjacent, as used herein, is where 2 nucleotides have no interveningnucleotides between them.

“Associated mutation,” as used herein, refers to a mutation within apreselected distance, in terms of nucleotide or primary amino acidsequence, from a definitional mutation, e.g., a mutant as describedherein. In embodiments, the associated mutation is within n, wherein nis 2, 5, 10, 20, 30, 50, 100, or 200 nucleotides from the definitionalmutation (n does not include the nucleotides defining the associated anddefinitional mutations). In embodiments, the associated mutation is atranslocation mutation.

“Interrogation position,” as used herein, comprises at least onenucleotide (or, in the case of polypeptides, an amino acid residue)which corresponds to a nucleotide (or amino acid residue) that ismutated in a mutation of interest, e.g., a mutation being identified, orin a nucleic acid (or protein) being analyzed, e.g., sequenced, orrecovered.

A “reference sequence,” as used herein, e.g., as a comparator for amutant sequence, is a sequence which has a different nucleotide or aminoacid at an interrogation position than does the mutant(s) beinganalyzed. In one embodiment, the reference sequence is wild-type for atleast the interrogation position.

Headings, e.g., (a), (b), (i) etc, are presented merely for ease ofreading the specification and claims. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

Various aspects featured in the invention are described in furtherdetail below. Additional definitions are set out throughout thespecification.

Therapeutic Methods and Agents

The invention provides, at least in part, methods for treating a cancer,e.g., a urothelial and/or micropapillary carcinoma (e.g., MPUC) in asubject. In certain embodiments, the methods include treatment of aurothelial and/or micropapillary carcinoma harboring an alterationdescribed herein (e.g., a HER2 alteration described herein). The methodsinclude administering to the subject a therapeutic agent, e.g., an agentthat antagonizes the function of HER2.

In certain embodiments, the cancer, e.g., the urothelial and/ormicropapillary carcinoma, is chosen from a cancer of the urinary system(e.g., kidney, bladder, ureter, urethra and urachus), urothelial cells,breast, lung, endometrium, bile duct or thyroid. In one embodiment, themicropapillary carcinoma is chosen from a cancer of the urinary tract,bladder, urothelial cells or bile duct. In one embodiment, themicropapillary carcinoma is a micropapillary urothelial carcinoma(MPUC). In one embodiment, the urothelial carcinoma is metastatic.

In certain embodiment, the cancer has, or is identified or determined ashaving, a histology of micropapillae. In certain embodiments, themicropapillary histology or histology of micropapillae comprises smallmicropapillae created by clusters of two or more cells (typically 4 to 5cells) across, peripherally situated nuclei and cytoplasmic vacuoles.

In other embodiment, the cancer, e.g., the urothelial and/ormicropapillary carcinoma (e.g., MPUC), comprises, or is identified ordetermined as having, an alteration in HER2, e.g., an alteration in HER2as described herein. In one embodiment, the urothelial and/ormicropapillary carcinoma (e.g., MPUC) does not have a gene amplificationor overexpression of HER2 or a HER2 gene product. For example, thecancer is not an ERBB2-amplified cancer or carcinoma (e.g., anERBB2-amplified urothelial carcinoma). In certain embodiments, thecancer does not have an elevated level of, or is negative for, a HER2gene product (e.g., the cancer is negative for HER2 expression bynucleic acid or protein detection methods, e.g., PCR orimmunohistochemistry).

“Treat,” “treatment,” and other forms of this word refer to theadministration of an agent, e.g., a therapeutic agent, alone or incombination with a second agent in an amount effective to impede growthof a cancer, to cause a cancer to shrink by weight or volume, to extendthe expected survival time of the subject and or time to progression ofthe tumor or the like. In those subjects, treatment can include, but isnot limited to, inhibiting tumor growth, reducing tumor mass, reducingsize or number of metastatic lesions, inhibiting the development of newmetastatic lesions, prolonged survival, prolonged progression-freesurvival, prolonged time to progression, and/or enhanced quality oflife. A cancer is “treated” if at least one symptom of the cancer isalleviated, terminated, slowed or prevented. A cancer is also “treated”if recurrence or metastasis of the cancer is reduced, slowed, delayed orprevented.

As used herein, unless otherwise specified, the terms “prevent,”“preventing” and “prevention” contemplate an action that occurs before asubject begins to suffer from the re-growth of the cancer and/or whichinhibits or reduces the severity of the cancer.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of an agent is an amount sufficient to provide atherapeutic benefit in the treatment or management of the cancer, or todelay or minimize one or more symptoms associated with the cancer. Atherapeutically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other therapeuticagents, which provides a therapeutic benefit in the treatment ormanagement of the cancer. The term “therapeutically effective amount”can encompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of the cancer, or enhances the therapeutic efficacyof another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of an agent is an amount sufficient to preventre-growth of the cancer, or one or more symptoms associated with thecancer, or prevent its recurrence. A prophylactically effective amountof an agent means an amount of the agent, alone or in combination withother therapeutic agents, which provides a prophylactic benefit in theprevention of the cancer. The term “prophylactically effective amount”can encompass an amount that improves overall prophylaxis or enhancesthe prophylactic efficacy of another prophylactic agent.

The term “patient” or “subject” includes a human (e g, a male or femaleof any age group, e.g., a pediatric patient (e.g., infant, child,adolescent) or adult patient (e.g., young adult, middle-aged adult orsenior adult). When the term is used in conjunction with administrationof a compound or drug, then the patient has been the object oftreatment, observation, and/or administration of the compound or drug.

These treatments can be provided to a patient having had anunsatisfactory response to a different (e.g., non-HER2) therapeuticagent or therapeutic modality. In one embodiment, the subject isundergoing or has undergone treatment with a different (e.g., non-HER2)therapeutic agent or therapeutic modality. In one embodiment, thenon-HER2 therapeutic agent or therapeutic modality is a chemotherapy ora surgical procedure. In one embodiment, the non-HER2 therapeutic agentor therapeutic modality comprises one or more of: methotrexate,vinblastine, doxorubicin, and/or cisplatin (MVAC).

An agent, e.g., therapeutic agent, described herein can be administered,alone or in combination, e.g., in combination with otherchemotherapeutic agents or procedures, in an amount sufficient to reduceor inhibit the tumor cell growth, and/or treat or prevent the cancer(s),in the subject.

The agent, e.g., therapeutic agent, can be a small molecule, a protein,a polypeptide, a peptide, an antibody molecule, a nucleic acid (e.g., asiRNA, an antisense or a micro RNA), a small molecule, or an immune celltherapy. Exemplary agents and classes of agents are described herein.

In one embodiment, the agent, e.g., therapeutic agent, binds andinhibits HER2. In one embodiment, the agent is an antibody molecule. Theterms “antibody” and “antibody molecule” as used interchangeably hereinrefer to immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as a polypeptidefeatured in the invention. A molecule which specifically binds to agiven polypeptide featured in the invention is a molecule which bindsthe polypeptide, but does not substantially bind other molecules in asample, e.g., a biological sample, which naturally contains thepolypeptide. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition,” as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. Antibodies to HER2 are known in the art, as well astechniques for generating antibodies to a polypeptide target, e.g., HER2(see e.g., WO 2012/092426, entitled “Optimization of Multigene Analysisof Tumor Samples,” incorporated herein by reference.

In another embodiment, the agent is selected from an antisense molecule,a ribozymes, a double-stranded RNA molecule, a triple helix molecule,that hybridize to a nucleic acid encoding the mutation, or atranscription regulatory region that blocks or reduces mRNA expressionof the mutation. In one embodiment, the agent is a kinase inhibitor. Inone embodiment, the kinase inhibitor is chosen from: a multi-specifickinase inhibitor, a HER2 inhibitor, an ERBB2 inhibitor, an EGFRinhibitor (e.g., a pan ERBB inhibitor), an antibody molecule, (e.g., amonoclonal antibody) against HER2, and/or a small molecule inhibitorthat is selective for HER2.

As used herein, a “pan ERBB inhibitor” is an inhibitor which is notspecific for one ERBB family member but can inhibit all ERBB familymembers, e.g., HER1, HER2, HER3, and HER4.

In one embodiment, the agent is chosen from: AV-203, AMG 888, U3-1287,APC8024, DN24-02, Neuvenge, Lapuleucel-T, MM-111, MM-121, SAR256212,MM-141, LJM716, REGN1400, MEHD7945A, RG7597, RG7116, Trastuzumab,trastuzumab emtansine (T-DM1), pertuzumab, afatinib, TAK-285, Neratinib,Dacomitinib, BMS-690514, BMS-599626, Pelitinib, CP-724714, Lapatinib,TAK-165, ARRY-380, or AZD8931. In one embodiment, the agent isNeratinib. Each of these inhibitors is described in more detail below.

AV-203 is a humanized monoclonal antibody directed against ErbB-3; andis described in Cancer Research: Apr. 15, 2012; Volume 72, Issue 8,Supplement.AM2012-2509.

AMG-888 (U3-1287) is a human monoclonal antibody directed againstepidermal growth factor receptor 3 (HER3); and is described in J ClinOncol 29: 2011 (suppl; abstr 3026).

APC 8024 (Lapuleucel-T, Neuvenge, DN24-02) is an investigationalautologous active cellular immunotherapy designed to stimulate an immuneresponse against tumor cells expressing the cancer antigen HER-2/neu;and is described in Clin Cancer Res Sep. 15, 2009 15; 5937.

MM-111 is a bispecific antibody fusion protein that specifically targetsthe ErbB2/ErbB3 heterodimer and abrogates ligand binding; and isdescribed in Cancer Res Dec. 15, 2010; 70(24 Supplement): P6-15-15.

MM-121 (sar256212) is a fully humanized monoclonal antibody directedagainst epidermal growth factor receptor 3 (HER3); and is described inCancer Res. 2010 Mar. 15; 70(6):2485-94.

MM-141 monoclonal antibody that acts as a tetravalent inhibitor ofPI3K/AKT/mTOR MM-141 is designed to interfere with this pathway byblocking ligand-induced signaling through the IGF-1R and ErbB3receptors; and is described in Oncotarget 2012 August; 3(8): 744-758.

LJM716 is a fully human HuCAL-based antibody directed against HER3; andis described in Cancer Research: Apr. 15, 2012; Volume 72, Issue 8,Supplement 1. AM2012-2733.

REGN1400 is a fully human monoclonal antibody directed against ERBB3;and is described in Cancer Research: Apr. 15, 2012; Volume 72, Issue 8,Supplement 1.

MEHD7945A (RG7597) is a monoclonal antibody that dually targets EGFR andHER3; and is described in Cancer Res Jan. 15, 2013 73; 824.

RG7116 is a glycoengineered humanized monoclonal antibody directedagainst HER3. RG7116 is a therapeutic antibody that binds the inactiveHER3 receptor and is optimized for immune effector activation; and isdescribed in Mirschberger C et al. Cancer Res. 2013 Jun. 18 [Epub aheadof print].

Trastuzumab is a monoclonal antibody directed against HER2; and isdescribed in British Journal of Cancer (2012) 106, 6-13.

Trastuzumab emtansine (T-DM1) is a monoclonal antibody directed againstHER2 conjugated to a cytotoxic moiety; and is described in BritishJournal of Cancer (2012) 106, 6-13.

Pertuzumab is a monoclonal antibody directed against HER2; and isdescribed in British Journal of Cancer (2012) 106, 6-13.

Afatinib is an anilino-quinazoline-derived irreversible small-moleculeinhibitor of the ErbB family (EGFR/HER1, HER2 and HER4); and isdescribed in British Journal of Cancer (2012) 106, 6-13.

TAK-285 is a low molecular weight compound which inhibits HER2 and EGFRkinase activities; and is described in Br J Cancer. 2012 Feb. 14;106(4).

Neratinib is an irreversible small-molecule inhibitor of EGFR/HER1, HER2and HER4; and is described in British Journal of Cancer (2012) 106,6-13.

Dacomitinib is an irreversible small molecule inhibitor of the pan-EGFRfamily of tyrosine kinases (ErbB family), including ErbB-1, ErbB-2, andErbB-3; and is described in PLoS One. 2013; 8(2): e56112.

BMS-690514 is a small molecule panHER/VEGFR/EGFR inhibitor; and isdescribed in Cancer Res. 2007 Jul. 1; 67(13):6253-62.

BMS-599626 is a pyrrolotriazine-based small-molecule panHER/EGFRinhibitor; and is described in Clin Cancer Res. 2006 Oct. 15; 12(20 Pt1):6186-93.

Pelitinib is a 3-cyanoquinoline pan-ErbB tyrosine kinase inhibitor,which irreversibly covalently binds to epidermal growth factor receptors(EGFR) ErbB-1, -2 and -4; and is described in Nature Reviews Cancer 10,760-774 (November 2010).

CP-724714 is a small molecule inhibitor of erbB2 receptor; and isdescribed in Cancer Res. 2007 Oct. 15; 67 (20):9887-93.

Lapatinib is a small molecule, reversible, dual inhibitor of EGFR/HER1and HER2; and is described in British Journal of Cancer (2012) 106,6-13.

TAK-165 (mubritinib) is a small molecule inhibitor of HER2; and isdescribed in Int J Urol. 2006 May; 13(5):587-92.

ARRY-380 is a small molecule reversible HER2 inhibitor; and is describedin Cancer Research: Dec. 15, 2009; Volume 69, Issue 24, Supplement 3.SABCS-09-5104.

AZD8931 is an equipotent reversible inhibitor of signaling by epidermalgrowth factor receptor, ERBB2 (HER2), and ERBB3; and is described inClin Cancer Res. 2010 Feb. 15; 16(4):1159-69.CCR-09-2353.

The agents, e.g., the therapeutic agents described herein, can beadministered in combination with a second therapeutic agent or adifferent therapeutic modality, e.g., anti-cancer agents, and/or incombination with surgical and/or radiation procedures.

By “in combination with,” it is not intended to imply that the therapyor the therapeutic agents must be administered at the same time and/orformulated for delivery together, although these methods of delivery arewithin the scope of the invention. The pharmaceutical compositions canbe administered concurrently with, prior to, or subsequent to, one ormore other additional therapies or therapeutic agents. In general, eachagent will be administered at a dose and/or on a time scheduledetermined for that agent. In will further be appreciated that theadditional therapeutic agent utilized in this combination can beadministered together in a single composition or administered separatelyin different compositions. The particular combination to employ in aregimen will take into account compatibility of the inventivepharmaceutical composition with the additional therapeutically activeagent and/or the desired therapeutic effect to be achieved.

In certain embodiments, the cancer, e.g., the micropapillary carcinoma,comprises, or is identified or determined as having, an alteration inone or more of: AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 or RAF1. In oneembodiment, the cancer has a wild-type HER2 and comprises an alterationin one or more of: AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 or RAF1. Suchcancers can be treated with modulators, e.g., inhibitors, of one or moreof: AKT1, AKT2, CCND1, EGFR, PIK3CA, PIK3R1 or RAF1.

Nucleic Acid Inhibitors

In yet another embodiment, the agent, e.g., the therapeutic agent,inhibits the expression of a nucleic acid encoding an alterationdescribed herein. Examples of such agents include nucleic acidmolecules, for example, antisense molecules, ribozymes, siRNA, triplehelix molecules that hybridize to a nucleic acid encoding a mutation, ora transcription regulatory region, and blocks or reduces mRNA expressionof the mutation.

In one embodiment, the nucleic acid antagonist is a siRNA that targetsmRNA encoding a mutation. Other types of antagonistic nucleic acids canalso be used, e.g., a dsRNA, a ribozyme, a triple-helix former, or anantisense nucleic acid. Accordingly, isolated nucleic acid moleculesthat are nucleic acid inhibitors, e.g., antisense, RNAi, to amutation-encoding nucleic acid molecule are provided.

An “antisense” nucleic acid can include a nucleotide sequence which iscomplementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. The antisense nucleic acid can becomplementary to an entire mutation coding strand, or to only a portionthereof. In another embodiment, the antisense nucleic acid molecule isantisense to a “noncoding region” of the coding strand of a nucleotidesequence encoding mutation (e.g., the 5′ and 3′ untranslated regions).Anti-sense agents can include, for example, from about 8 to about 80nucleobases (i.e., from about 8 to about 80 nucleotides), e.g., about 8to about 50 nucleobases, or about 12 to about 30 nucleobases. Anti-sensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression. Anti-sense compounds can include a stretchof at least eight consecutive nucleobases that are complementary to asequence in the target gene. An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA can interfere withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all key functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

Exemplary antisense compounds include DNA or RNA sequences thatspecifically hybridize to the target nucleic acid, e.g., the mRNAencoding a mutation described herein. The complementary region canextend for between about 8 to about 80 nucleobases. The compounds caninclude one or more modified nucleobases. Modified nucleobases are knownin the art. Descriptions of modified nucleic acid agents are alsoavailable. See, e.g., U.S. Pat. Nos. 4,987,071; 5,116,742; and U.S. Pat.No. 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci USA; Antisense RNAand DNA, D. A. Melton, Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature334:585-59; Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene(1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14:807-15.

The antisense nucleic acid molecules are typically administered to asubject (e.g., by direct injection at a tissue site), or generated insitu such that they hybridize with or bind to cellular mRNA and/orgenomic DNA encoding a mutation to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then be administered systemically. Forsystemic administration, antisense molecules can be modified such thatthey specifically bind to receptors or antigens expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule is anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

siRNAs are small double stranded RNAs (dsRNAs) that optionally includeoverhangs. For example, the duplex region of an siRNA is about 18 to 25nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotidesin length. Typically, the siRNA sequences are exactly complementary tothe target mRNA. dsRNAs and siRNAs in particular can be used to silencegene expression in mammalian cells (e.g., human cells). siRNAs alsoinclude short hairpin RNAs (shRNAs) with 29-base-pair stems and2-nucleotide 3′ overhangs. See, e.g., Clemens et al. (2000) Proc. Natl.Acad. Sci. USA 97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA98:14428-14433; Elbashir et al. (2001) Nature. 411:494-8; Yang et al.(2002) Proc. Natl. Acad. Sci. USA 99:9942-9947; Siolas et al. (2005),Nat. Biotechnol. 23(2):227-31; 20040086884; U.S. 20030166282;20030143204; 20040038278; and 20030224432.

In still another embodiment, an antisense nucleic acid featured in theinvention is a ribozyme. A ribozyme having specificity for amutation-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a mutation cDNA disclosedherein (i.e., SEQ ID NO:6), and a sequence having known catalyticsequence responsible for mRNA cleavage (see U.S. Pat. No. 5,093,246 orHaselhoff and Gerlach (1988) Nature 334:585-591). For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a mutation-encoding mRNA. See,e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No.5,116,742. Alternatively, mutation mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

Inhibition of a mutated gene can be accomplished by targeting nucleotidesequences complementary to the regulatory region of the mutation to formtriple helical structures that prevent transcription of the mutated genein target cells. See generally, Helene, C. (1991) Anticancer Drug Des.6:569-84; Helene, C. i (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,L. J. (1992) Bioassays 14:807-15. The potential sequences that can betargeted for triple helix formation can be increased by creating aso-called “switchback” nucleic acid molecule. Switchback molecules aresynthesized in an alternating 5′-3′, 3′-5′ manner, such that they basepair with first one strand of a duplex and then the other, eliminatingthe necessity for a sizeable stretch of either purines or pyrimidines tobe present on one strand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A mutated nucleic acid molecule can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For non-limiting examplesof synthetic oligonucleotides with modifications see Toulmé (2001)Nature Biotech. 19:17 and Faria et al. (2001) Nature Biotech. 19:40-44.Such phosphoramidite oligonucleotides can be effective antisense agents.

For example, the deoxyribose phosphate backbone of the nucleic acidmolecules can be modified to generate peptide nucleic acids (see HyrupB. et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23). As usedherein, the terms “peptide nucleic acid” or “PNA” refers to a nucleicacid mimic, e.g., a DNA mimic, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of a PNA canallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in HyrupB. et al. (1996) supra and Perry-O'Keefe et al. Proc. Natl. Acad. Sci.93: 14670-675.

PNAs of mutated nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of mutated nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., 51 nucleases (Hyrup B. etal. (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652;WO88/09810) or the blood-brain barrier (see, e.g., WO 89/10134). Inaddition, oligonucleotides can be modified with hybridization-triggeredcleavage agents (see, e.g., Krol et al. (1988) Bio-Techniques 6:958-976)or intercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549).To this end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

In some embodiments, a nucleic acid inhibitor described herein isprovided for the inhibition of expression of a nucleic acid comprisingthe alteration in vitro.

Evaluation of Subjects

Subjects, e.g., patients, can be evaluated for the presence of analteration, e.g., an alteration as described herein. A patient can beevaluated, for example, by determining the genomic sequence of thepatient, e.g., by an NGS method. Alternatively, or in addition,evaluation of a patient can include directly assaying for the presenceof a mutation in the patient, such as by an assay to detect a mutatednucleic acid (e.g., DNA or RNA), such as by, Southern blot, Northernblot, or RT-PCR, e.g., qRT-PCR. Alternatively, or in addition, a patientcan be evaluated for the presence of a protein mutation, such as byimmunohistochemistry, Western blot, immunoprecipitation, orimmunomagnetic bead assay.

In one aspect, the results of a clinical trial, e.g., a successful orunsuccessful clinical trial, can be repurposed to identify agents thattarget an alteration disclosed herein, e.g., a HER2 mutation. By oneexemplary method, a candidate agent used in a clinical trial can bereevaluated to determine if the agent in the trial targets a mutation,or is effective to treat a tumor containing a particular mutation. Forexample, subjects who participated in a clinical trial for an agent,such as a kinase inhibitor, can be identified. Patients who experiencedan improvement in symptoms, e.g., cancer (e.g., a urothelial and/ormicropapillary carcinoma, e.g., MPUC) symptoms, such as decreased tumorsize, or decreased rate of tumor growth, can be evaluated for thepresence of a mutation. Patients who did not experience an improvementin cancer symptoms can also be evaluated for the presence of a mutation.Where patients carrying a mutation are found to have been more likely torespond to the test agent than patients who did not carry such amutation, then the agent is determined to be an appropriate treatmentoption for a patient carrying the mutation.

“Reevaluation” of patients can include, for example, determining thegenomic sequence of the patients, or a subset of the clinical trialpatients, e.g., by an NGS method. Alternatively, or in addition,reevaluation of the patients can include directly assaying for thepresence of a mutation in the patient, such as by an assay to detect amutated nucleic acid (e.g., RNA), such as by RT-PCR, e.g., qRT-PCR.Alternatively, or in addition, a patient can be evaluated for thepresence of a protein mutation, such as by immunohistochemistry, Westernblot, immunoprecipitation, or immunomagnetic bead assay.

Methods for Detection of Nucleic Acids and Polypeptides

Methods for evaluating a mutated gene, mutations and/or gene productsare known to those of skill in the art. In one embodiment, the mutationis detected in a nucleic acid molecule by a method chosen from one ormore of: nucleic acid hybridization assay, SSP, HPLC ormass-spectrometric genotyping.

Additional exemplary methods include traditional “direct probe” methodssuch as Southern blots and “comparative probe” methods such ascomparative genomic hybridization (CGH), e.g., cDNA-based oroligonucleotide-based CGH, can be used. The methods can be used in awide variety of formats including, but not limited to, substrate (e.g.,membrane or glass) bound methods or array-based approaches.

In certain embodiments, the evaluation methods include probes/primersagainst the alterations described herein.

In one embodiment, probes/primers can be designed to detect a mutationor a reciprocal thereof. These probes/primers are suitable, e.g., forPCR amplification. Probes are used that contain DNA segments that areessentially complementary to DNA base sequences existing in differentportions of chromosomes. Examples of probes useful according to theinvention, and labeling and hybridization of probes to samples aredescribed in two U.S. patents to Vysis, Inc. U.S. Pat. Nos. 5,491,224and 6,277,569 to Bittner, et al.

Chromosomal probes are typically about 50 to about 10⁵ nucleotides inlength. Longer probes typically comprise smaller fragments of about 100to about 500 nucleotides in length. Probes that hybridize withcentromeric DNA and locus-specific DNA are available commercially, forexample, from Vysis, Inc. (Downers Grove, Ill.), Molecular Probes, Inc.(Eugene, Oreg.) or from Cytocell (Oxfordshire, UK). Alternatively,probes can be made non-commercially from chromosomal or genomic DNAthrough standard techniques. For example, sources of DNA that can beused include genomic DNA, cloned DNA sequences, somatic cell hybridsthat contain one, or a part of one, chromosome (e.g., human chromosome)along with the normal chromosome complement of the host, and chromosomespurified by flow cytometry or microdissection. The region of interestcan be isolated through cloning, or by site-specific amplification viathe polymerase chain reaction (PCR). See, for example, Nath and Johnson,Biotechnic Histochem., 1998, 73(1):6-22, Wheeless et al., Cytometry1994, 17:319-326, and U.S. Pat. No. 5,491,224.

The probes to be used hybridize to a specific region of a chromosome todetermine whether a cytogenetic abnormality is present in this region.One type of cytogenetic abnormality is a deletion. Although deletionscan be of one or more entire chromosomes, deletions normally involveloss of part of one or more chromosomes. If the entire region of achromosome that is contained in a probe is deleted from a cell,hybridization of that probe to the DNA from the cell will normally notoccur and no signal will be present on that chromosome. If the region ofa chromosome that is partially contained within a probe is deleted froma cell, hybridization of that probe to the DNA from the cell can stilloccur, but less of a signal can be present. For example, the loss of asignal is compared to probe hybridization to DNA from control cells thatdo not contain the genetic abnormalities which the probes are intendedto detect. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, ormore cells are enumerated for presence of the cytogenetic abnormality.

Cytogenetic abnormalities to be detected can include, but are notlimited to, non-reciprocal translocations, balanced translocations,intra-chromosomal inversions, point mutations, deletions, gene copynumber changes, gene expression level changes, and germ line mutations.In particular, one type of cytogenetic abnormality is a duplication.Duplications can be of entire chromosomes, or of regions smaller than anentire chromosome. If the region of a chromosome that is contained in aprobe is duplicated in a cell, hybridization of that probe to the DNAfrom the cell will normally produce at least one additional signal ascompared to the number of signals present in control cells with noabnormality of the chromosomal region contained in the probe.

Chromosomal probes are labeled so that the chromosomal region to whichthey hybridize can be detected. Probes typically are directly labeledwith a fluorophore, an organic molecule that fluoresces after absorbinglight of lower wavelength/higher energy. The fluorophore allows theprobe to be visualized without a secondary detection molecule. Aftercovalently attaching a fluorophore to a nucleotide, the nucleotide canbe directly incorporated into the probe with standard techniques such asnick translation, random priming, and PCR labeling. Alternatively,deoxycytidine nucleotides within the probe can be transaminated with alinker. The fluorophore then is covalently attached to the transaminateddeoxycytidine nucleotides. See, U.S. Pat. No. 5,491,224.

U.S. Pat. No. 5,491,224 describes probe labeling as a number of thecytosine residues having a fluorescent label covalently bonded thereto.The number of fluorescently labeled cytosine bases is sufficient togenerate a detectable fluorescent signal while the individual so labeledDNA segments essentially retain their specific complementary binding(hybridizing) properties with respect to the chromosome or chromosomeregion to be detected. Such probes are made by taking the unlabeled DNAprobe segment, transaminating with a linking group a number ofdeoxycytidine nucleotides in the segment, covalently bonding afluorescent label to at least a portion of the transaminateddeoxycytidine bases.

Probes can also be labeled by nick translation, random primer labelingor PCR labeling. Labeling is done using either fluorescent (direct)- orhaptene (indirect)-labeled nucleotides. Representative, non-limitingexamples of labels include: AMCA-6-dUTP, CascadeBlue-4-dUTP,Fluorescein-12-dUTP, Rhodamine-6-dUTP, TexasRed-6-dUTP, Cy3-6-dUTP,Cy5-dUTP, Biotin(BIO)-11-dUTP, Digoxygenin(DIG)-11-dUTP or Dinitrophenyl(DNP)-11-dUTP.

Probes also can be indirectly labeled with biotin or digoxygenin, orlabeled with radioactive isotopes such as ³²P and ³H, although secondarydetection molecules or further processing then is required to visualizethe probes. For example, a probe labeled with biotin can be detected byavidin conjugated to a detectable marker. For example, avidin can beconjugated to an enzymatic marker such as alkaline phosphatase orhorseradish peroxidase. Enzymatic markers can be detected in standardcolorimetric reactions using a substrate and/or a catalyst for theenzyme. Catalysts for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a catalyst for horseradish peroxidase.

Probes can also be prepared such that a fluorescent or other label isnot part of the DNA before or during the hybridization, and is addedafter hybridization to detect the probe hybridized to a chromosome. Forexample, probes can be used that have antigenic molecules incorporatedinto the DNA. After hybridization, these antigenic molecules aredetected using specific antibodies reactive with the antigenicmolecules. Such antibodies can themselves incorporate a fluorochrome, orcan be detected using a second antibody with a bound fluorochrome.

However treated or modified, the probe DNA is commonly purified in orderto remove unreacted, residual products (e.g., fluorochrome molecules notincorporated into the DNA) before use in hybridization.

Prior to hybridization, chromosomal probes are denatured according tomethods well known in the art. Probes can be hybridized or annealed tothe chromosomal DNA under hybridizing conditions. “Hybridizingconditions” are conditions that facilitate annealing between a probe andtarget chromosomal DNA. Since annealing of different probes will varydepending on probe length, base concentration and the like, annealing isfacilitated by varying probe concentration, hybridization temperature,salt concentration and other factors well known in the art.

Hybridization conditions are facilitated by varying the concentrations,base compositions, complexities, and lengths of the probes, as well assalt concentrations, temperatures, and length of incubation. Forexample, in situ hybridizations are typically performed in hybridizationbuffer containing 1-2×SSC, 50-65% formamide and blocking DNA to suppressnon-specific hybridization. In general, hybridization conditions, asdescribed above, include temperatures of about 25° C. to about 55° C.,and incubation lengths of about 0.5 hours to about 96 hours.

Non-specific binding of chromosomal probes to DNA outside of the targetregion can be removed by a series of washes. Temperature andconcentration of salt in each wash are varied to control stringency ofthe washes. For example, for high stringency conditions, washes can becarried out at about 65° C. to about 80° C., using 0.2× to about 2×SSC,and about 0.1% to about 1% of a non-ionic detergent such as Nonidet P-40(NP40). Stringency can be lowered by decreasing the temperature of thewashes or by increasing the concentration of salt in the washes. In someapplications it is necessary to block the hybridization capacity ofrepetitive sequences. Thus, in some embodiments, tRNA, human genomicDNA, or Cot-I DNA is used to block non-specific hybridization. Afterwashing, the slide is allowed to drain and air dry, then mountingmedium, a counterstain such as DAPI, and a coverslip are applied to theslide. Slides can be viewed immediately or stored at −20° C. beforeexamination.

In CGH methods, a first collection of nucleic acids (e.g., from asample, e.g., a possible tumor) is labeled with a first label, while asecond collection of nucleic acids (e.g., a control, e.g., from ahealthy cell/tissue) is labeled with a second label. The ratio ofhybridization of the nucleic acids is determined by the ratio of the two(first and second) labels binding to each fiber in the array. Wherethere are chromosomal deletions or multiplications, differences in theratio of the signals from the two labels will be detected and the ratiowill provide a measure of the copy number. Array-based CGH can also beperformed with single-color labeling (as opposed to labeling the controland the possible tumor sample with two different dyes and mixing themprior to hybridization, which will yield a ratio due to competitivehybridization of probes on the arrays). In single color CGH, the controlis labeled and hybridized to one array and absolute signals are read,and the possible tumor sample is labeled and hybridized to a secondarray (with identical content) and absolute signals are read. Copynumber difference is calculated based on absolute signals from the twoarrays. Hybridization protocols suitable for use with the methodsfeatured in the invention are described, e.g., in Albertson (1984) EMBOJ. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142;EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situHybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994),etc. In one embodiment, the hybridization protocol of Pinkel, et al.(1998) Nature Genetics 20: 207-211, or of Kallioniemi (1992) Proc. NatlAcad Sci USA 89:5321-5325 (1992) is used. Array-based CGH is describedin U.S. Pat. No. 6,455,258, the contents of each of which areincorporated herein by reference.

In still another embodiment, amplification-based assays can be used tomeasure presence/absence and copy number. In such amplification-basedassays, the nucleic acid sequences act as a template in an amplificationreaction (e.g., Polymerase Chain Reaction (PCR). In a quantitativeamplification, the amount of amplification product will be proportionalto the amount of template in the original sample. Comparison toappropriate controls, e.g., healthy tissue, provides a measure of thecopy number.

Methods of “quantitative” amplification are well known to those of skillin the art. For example, quantitative PCR involves simultaneouslyco-amplifying a known quantity of a control sequence using the sameprimers. This provides an internal standard that can be used tocalibrate the PCR reaction. Detailed protocols for quantitative PCR areprovided in Innis, et al. (1990) PCR Protocols, A Guide to Methods andApplications, Academic Press, Inc. N.Y.). Measurement of DNA copy numberat microsatellite loci using quantitative PCR analysis is described inGinzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleicacid sequence for the genes is sufficient to enable one of skill in theart to routinely select primers to amplify any portion of the gene.Fluorogenic quantitative PCR can also be used. In fluorogenicquantitative PCR, quantitation is based on amount of fluorescencesignals, e.g., TaqMan and sybr green.

Other suitable amplification methods include, but are not limited to,ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990)Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc.Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication(Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR,and linker adapter PCR, etc.

Nucleic Acid Samples

A variety of tissue samples can be the source of the nucleic acidsamples used in the present methods. Genomic or subgenomic DNA fragmentscan be isolated from a subject's sample (e.g., a tumor sample, a normaladjacent tissue (NAT), a blood sample or any normal control)). Incertain embodiments, the tissue sample is preserved as a frozen sampleor as formaldehyde- or paraformaldehyde-fixed paraffin-embedded (FFPE)tissue preparation. For example, the sample can be embedded in a matrix,e.g., an FFPE block or a frozen sample. The isolating step can includeflow-sorting of individual chromosomes; and/or micro-dissecting asubject's sample (e.g., a tumor sample, a NAT, a blood sample).

Protocols for DNA isolation, fragmentation and processing from a tissuesample are known in the art as described, e.g., in WO 2012/092426,entitled “Optimization of Multigene Analysis of Tumor Samples,”incorporated herein by reference in its entirety. Additional methods toisolate nucleic acids (e.g., DNA) from formaldehyde- orparaformaldehyde-fixed, paraffin-embedded (FFPE) tissues are disclosed,e.g., in Cronin M. et al., (2004) Am J Pathol. 164(1):35-42; Masuda N.et al., (1999) Nucleic Acids Res. 27(22):4436-4443; Specht K. et al.,(2001) Am J Pathol. 158(2):419-429, Ambion RecoverAll™ Total NucleicAcid Isolation Protocol (Ambion, Cat. No. AM1975, September 2008), andQIAamp® DNA FFPE Tissue Handbook (Qiagen, Cat. No. 37625, October 2007).RecoverAll™ Total Nucleic Acid Isolation Kit uses xylene at elevatedtemperatures to solubilize paraffin-embedded samples and a glass-fiberfilter to capture nucleic acids. QIAamp® DNA FFPE Tissue Kit usesQIAamp® DNA Micro technology for purification of genomic andmitochondrial DNA.

Design of Baits

A bait can be a nucleic acid molecule, e.g., a DNA or RNA molecule,which can hybridize to (e.g., be complementary to), and thereby allowcapture of a target nucleic acid. In one embodiment, a bait is an RNAmolecule. In other embodiments, a bait includes a binding entity, e.g.,an affinity tag, that allows capture and separation, e.g., by binding toa binding entity, of a hybrid formed by a bait and a nucleic acidhybridized to the bait. In one embodiment, a bait is suitable forsolution phase hybridization.

Baits can be produced and used by methods and hybridization conditionsas described in US 2010/0029498 and Gnirke, A. et al. (2009) NatBiotechnol. 27(2):182-189, and WO 2012/092426, entitled “Optimization ofMultigene Analysis of Tumor Samples, incorporated herein by reference.

Sequencing

The invention also includes methods of sequencing nucleic acids. In oneembodiment, any of a variety of sequencing reactions known in the artcan be used to directly sequence at least a portion of a mutation. Inone embodiment, the mutated sequence is compared to a correspondingreference (control) sequence.

In one embodiment, the sequence of the nucleic acid molecule comprisingan alteration described herein is determined by a method that includesone or more of: hybridizing an oligonucleotide, e.g., an allele specificoligonucleotide for one mutation described herein to said nucleic acid;hybridizing a primer, or a primer set (e.g., a primer pair), thatamplifies a region comprising the mutation of the allele; amplifying,e.g., specifically amplifying, a region comprising the mutation of theallele; attaching an adapter oligonucleotide to one end of a nucleicacid that comprises the mutation of the allele; generating an optical,e.g., a colorimetric signal, specific to the presence of the one of themutation; hybridizing a nucleic acid comprising the mutation to a secondnucleic acid, e.g., a second nucleic acid attached to a substrate;generating a signal, e.g., an electrical or fluorescent signal, specificto the presence of the mutation; and incorporating a nucleotide into anoligonucleotide that is hybridized to a nucleic acid that contains themutation.

In another embodiment, the sequence is determined by a method thatcomprises one or more of: determining the nucleotide sequence from anindividual nucleic acid molecule, e.g., where a signal corresponding tothe sequence is derived from a single molecule as opposed, e.g., from asum of signals from a plurality of clonally expanded molecules;determining the nucleotide sequence of clonally expanded proxies forindividual nucleic acid molecules; massively parallel short-readsequencing; template-based sequencing; pyrosequencing; real-timesequencing comprising imaging the continuous incorporation ofdye-labeling nucleotides during DNA synthesis; nanopore sequencing;sequencing by hybridization; nano-transistor array based sequencing;polony sequencing; scanning tunneling microscopy (STM) based sequencing;or nanowire-molecule sensor based sequencing.

Any method of sequencing known in the art can be used. Exemplarysequencing reactions include those based on techniques developed byMaxam and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger(Sanger et al. (1977) Proc. Nat. Acad. Sci 74:5463). Any of a variety ofautomated sequencing procedures can be utilized when performing theassays (Biotechniques (1995) 19:448), including sequencing by massspectrometry (see, for example, U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/16101,entitled DNA Sequencing by Mass Spectrometry by H. Köster; U.S. Pat. No.5,547,835 and international patent application Publication Number WO94/21822 entitled DNA Sequencing by Mass Spectrometry Via ExonucleaseDegradation by H. Köster), and U.S. Pat. No. 5,605,798 and InternationalPatent Application No. PCT/US96/03651 entitled DNA Diagnostics Based onMass Spectrometry by H. Köster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159).

Sequencing of nucleic acid molecules can also be carried out usingnext-generation sequencing (NGS). Next-generation sequencing includesany sequencing method that determines the nucleotide sequence of eitherindividual nucleic acid molecules or clonally expanded proxies forindividual nucleic acid molecules in a highly parallel fashion (e.g.,greater than 10⁵ molecules are sequenced simultaneously). In oneembodiment, the relative abundance of the nucleic acid species in thelibrary can be estimated by counting the relative number of occurrencesof their cognate sequences in the data generated by the sequencingexperiment. Next generation sequencing methods are known in the art, andare described, e.g., in Metzker, M. (2010) Nature Biotechnology Reviews11:31-46, incorporated herein by reference.

In one embodiment, the next-generation sequencing allows for thedetermination of the nucleotide sequence of an individual nucleic acidmolecule (e.g., Helicos BioSciences' HeliScope Gene Sequencing system,and Pacific Biosciences' PacBio RS system). In other embodiments, thesequencing method determines the nucleotide sequence of clonallyexpanded proxies for individual nucleic acid molecules (e.g., the Solexasequencer, Illumina Inc., San Diego, Calif.; 454 Life Sciences(Branford, Conn.), and Ion Torrent). e.g., massively parallel short-readsequencing (e.g., the Solexa sequencer, Illumina Inc., San Diego,Calif.), which generates more bases of sequence per sequencing unit thanother sequencing methods that generate fewer but longer reads. Othermethods or machines for next-generation sequencing include, but are notlimited to, the sequencers provided by 454 Life Sciences (Branford,Conn.), Applied Biosystems (Foster City, Calif.; SOLiD sequencer), andHelicos BioSciences Corporation (Cambridge, Mass.).

Platforms for next-generation sequencing include, but are not limitedto, Roche/454's Genome Sequencer (GS) FLX System, Illumina/Solexa'sGenome Analyzer (GA), Life/APG's Support Oligonucleotide LigationDetection (SOLiD) system, Polonator's G.007 system, Helicos BioSciences'HeliScope Gene Sequencing system, and Pacific Biosciences' PacBio RSsystem.

NGS technologies can include one or more of steps, e.g., templatepreparation, sequencing and imaging, and data analysis as described inWO 2012/092426, entitled “Optimization of Multigene Analysis of TumorSamples, incorporated herein by reference.

Data Analysis

After NGS reads have been generated, they can be aligned to a knownreference sequence or assembled de novo.

For example, identifying genetic variations such as single-nucleotidepolymorphism and structural variants in a sample (e.g., a tumor sample)can be accomplished by aligning NGS reads to a reference sequence (e.g.,a wild-type sequence). Methods of sequence alignment for NGS aredescribed e.g., in Trapnell C. and Salzberg S. L. Nature Biotech., 2009,27:455-457.

Examples of de novo assemblies are described, e.g., in Warren R. et al.,Bioinformatics, 2007, 23:500-501; Butler J. et al., Genome Res., 2008,18:810-820; and Zerbino D. R. and Birney E., Genome Res., 2008,18:821-829.

Sequence alignment or assembly can be performed using read data from oneor more NGS platforms, e.g., mixing Roche/454 and Illumina/Solexa readdata.

Algorithms and methods for data analysis are described in WO2012/092426, entitled “Optimization of Multigene Analysis of TumorSamples, incorporated herein by reference.

Detection of Mutated Polypeptide

The activity or level of a mutated polypeptide (e.g., a HER2 mutation)can also be detected and/or quantified by detecting or quantifying theexpressed polypeptide. The mutated polypeptide can be detected andquantified by any of a number of means known to those of skill in theart. These can include analytic biochemical methods such aselectrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, or various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, Westernblotting, immunohistochemistry (IHC) and the like. A skilled artisan canadapt known protein/antibody detection methods.

Another agent for detecting a mutated polypeptide is an antibodymolecule capable of binding to a polypeptide corresponding to apolypeptide, e.g., an antibody with a detectable label. Techniques forgenerating antibodies are described herein. The term “labeled”, withregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin.

In another embodiment, the antibody is labeled, e.g., a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody. Inanother embodiment, an antibody derivative (e.g., an antibody conjugatedwith a substrate or with the protein or ligand of a protein-ligand pair{e.g., biotin-streptavidin}), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a mutated protein, is used.

Mutated polypeptides from cells can be isolated using techniques thatare known to those of skill in the art. The protein isolation methodsemployed can, for example, be such as those described in Harlow and Lane(Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York).

Means of detecting proteins using electrophoretic techniques are wellknown to those of skill in the art (see generally, R. Scopes (1982)Protein Purification, Springer-Verlag, N.Y.; Deutscher, (1990) Methodsin Enzymology Vol. 182: Guide to Protein Purification, Academic Press,Inc., N.Y.).

In another embodiment, Western blot (immunoblot) analysis is used todetect and quantify the presence of a polypeptide in the sample.

In another embodiment, the polypeptide is detected using an immunoassay.As used herein, an immunoassay is an assay that utilizes an antibody tospecifically bind to the analyte. The immunoassay is thus characterizedby detection of specific binding of a polypeptide to an anti-antibody asopposed to the use of other physical or chemical properties to isolate,target, and quantify the analyte.

The mutated polypeptide is detected and/or quantified using any of anumber of immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Asai (1993) Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Academic Press, Inc. New York;Stites & Terr (1991) Basic and Clinical Immunology 7th Edition.

Kits

In one aspect, the invention features, a kit, e.g., containing anoligonucleotide having an alteration described herein, e.g., a HER2mutation. Optionally, the kit can also contain an oligonucleotide thatis the wildtype counterpart of the mutant oligonucleotide.

A kit can include a carrier, e.g., a means being compartmentalized toreceive in close confinement one or more container means. In oneembodiment the container contains an oligonucleotide, e.g., a primer orprobe as described above. The components of the kit are useful, forexample, to diagnose or identify a mutation in a tumor sample in apatient. The probe or primer of the kit can be used in any sequencing ornucleotide detection assay known in the art, e.g., a sequencing assay,e.g., an NGS method, RT-PCR, or in situ hybridization.

In some embodiments, the components of the kit are useful, for example,to diagnose or identify a mutation in a tumor sample in a patient, andto accordingly identify an appropriate therapeutic agent to treat thecancer.

A kit featured in the invention can include, e.g., assay positive andnegative controls, nucleotides, enzymes (e.g., RNA or DNA polymerase orligase), solvents or buffers, a stabilizer, a preservative, a secondaryantibody, e.g., an anti-HRP antibody (IgG) and a detection reagent.

An oligonucleotide can be provided in any form, e.g., liquid, dried,semi-dried, or lyophilized, or in a form for storage in a frozencondition.

Typically, an oligonucleotide, and other components in a kit areprovided in a form that is sterile. An oligonucleotide, e.g., anoligonucleotide that contains a mutation, described herein, or anoligonucleotide complementary to an alteration described herein, isprovided in a liquid solution, the liquid solution generally is anaqueous solution, e.g., a sterile aqueous solution. When theoligonucleotide is provided as a dried form, reconstitution generally isaccomplished by the addition of a suitable solvent. The solvent, e.g.,sterile buffer, can optionally be provided in the kit.

The kit can include one or more containers for the compositioncontaining an oligonucleotide in a concentration suitable for use in theassay or with instructions for dilution for use in the assay. In someembodiments, the kit contains separate containers, dividers orcompartments for the oligonucleotide and assay components, and theinformational material. For example, the oligonucleotides can becontained in a bottle or vial, and the informational material can becontained in a plastic sleeve or packet. In other embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, an oligonucleotide composition is contained in abottle or vial that has attached thereto the informational material inthe form of a label. In some embodiments, the kit includes a plurality(e.g., a pack) of individual containers, each containing one or moreunit forms (e.g., for use with one assay) of an oligonucleotide. Forexample, the kit includes a plurality of ampoules, foil packets, orblister packs, each containing a single unit of oligonucleotide for usein sequencing or detecting a mutation in a tumor sample. The containersof the kits can be air tight and/or waterproof. The container can belabeled for use.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a mutatedpolypeptide; and, optionally, (2) a second, different antibody whichbinds to either the polypeptide or the first antibody and is conjugatedto a detectable agent.

In one embodiment, the kit can include informational material forperforming and interpreting the sequencing or diagnostic. In anotherembodiment, the kit can provide guidance as to where to report theresults of the assay, e.g., to a treatment center or healthcareprovider. The kit can include forms for reporting the results of asequencing or diagnostic assay described herein, and address and contactinformation regarding where to send such forms or other relatedinformation; or a URL (Uniform Resource Locator) address for reportingthe results in an online database or an online application (e.g., anapp). In another embodiment, the informational material can includeguidance regarding whether a patient should receive treatment with aparticular chemotherapeutic drug, depending on the results of the assay.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawings, and/or photographs,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about the sequencingor diagnostic assay and/or its use in the methods described herein. Theinformational material can also be provided in any combination offormats.

In some embodiments, a biological sample is provided to an assayprovider, e.g., a service provider (such as a third party facility) or ahealthcare provider, who evaluates the sample in an assay and provides aread out. For example, in one embodiment, an assay provider receives abiological sample from a subject, such as a blood or tissue sample,e.g., a biopsy sample, and evaluates the sample using an assay describedherein, e.g., a sequencing assay or in situ hybridization assay, anddetermines that the sample contains a mutation. The assay provider,e.g., a service provider or healthcare provider, can then conclude thatthe subject is, or is not, a candidate for a particular drug or aparticular cancer treatment regimen.

Other embodiments of the invention include the following.

Nucleic Acid Molecules, Detection and Capturing Reagents

The invention also features an isolated nucleic acid molecule, or anisolated preparation of nucleic acid molecules, that includes analteration described herein. Such nucleic acid molecules or preparationsthereof can include an alteration described herein or can be used todetect, e.g., sequence, an alteration.

The invention also features a nucleic acid molecule, e.g., nucleic acidfragment, suitable as probe, primer, bait or library member thatincludes, flanks, hybridizes to, which are useful for identifying, orare otherwise based on, an alteration described herein. In certainembodiments, the probe, primer or bait molecule is an oligonucleotidethat allows capture, detection or isolation of a nucleic acid moleculecontaining an alteration described herein, e.g., an alteration in ERBB2,e.g. a mutation in HER2 at residue 5310 in which residue 5310 mutated toS310F or S310Y; or a mutation at residue 157 of HER2.

The oligonucleotide can comprise a nucleotide sequence substantiallycomplementary to nucleic acid molecules or fragments of nucleic acidmolecules comprising an alteration described herein. The sequenceidentity between the nucleic acid molecule, e.g., the oligonucleotide,and the target sequence need not be exact, so long as the sequences aresufficiently complementary to allow the capture, detection or isolationof the target sequence. In one embodiment, the nucleic acid fragment isa probe or primer that includes an oligonucleotide between about 5 and25, e.g., between 10 and 20, or 10 and 15 nucleotides in length. Inother embodiments, the nucleic acid fragment is a bait that includes anoligonucleotide between about 100 to 300 nucleotides, 130 and 230nucleotides, or 150 and 200 nucleotides, in length.

In one embodiment, the nucleic acid fragment can be used to identify orcapture, e.g., by hybridization, a nucleic acid molecules comprising analteration described herein, e.g., an alteration in HER2, e.g., amutation in HER2 at residue 5310 in which residue 5310 mutated to S310For S310Y; or a mutation at residue 157 of HER2. For example, the nucleicacid fragment can be a probe, a primer, or a bait, for use inidentifying or capturing, e.g., by hybridization, an alterationdescribed herein.

The probes or primers described herein can be used, for example, PCRamplification. In one exemplary embodiment where detection is based onPCR, amplification of the mutation can be performed using a primer or aprimer pair, e.g., for amplifying a sequence flanking an alterationdescribed herein.

In other embodiments, the nucleic acid fragment includes a bait thatcomprises a nucleotide sequence that hybridizes to a nucleic acidmolecules comprising an alteration described herein, and thereby allowsthe capture or isolation said nucleic acid molecule. In one embodiment,a bait is suitable for solution phase hybridization. In otherembodiments, a bait includes a binding entity, e.g., an affinity tag,that allows capture and separation, e.g., by binding to a bindingentity, of a hybrid formed by a bait and a nucleic acid hybridized tothe bait.

In other embodiments, the nucleic acid fragment includes a librarymember comprising a nucleic acid molecule described herein. In oneembodiment, the library member includes a mutation, e.g., a basesubstitution, that results in an alteration described herein.

The nucleic acid fragment can be detectably labeled with, e.g., aradiolabel, a fluorescent label, a bioluminescent label, achemiluminescent label, an enzyme label, a binding pair label, or caninclude an affinity tag; a tag, or identifier (e.g., an adaptor, barcodeor other sequence identifier).

Polypeptides

In another aspect, the disclosure features a polypeptide comprising analteration described herein (e.g., a purified polypeptide comprising analteration described herein), a biologically active or antigenicfragment thereof, as well as reagents (e.g., antibody molecules thatbind to a polypeptide comprising an alteration described herein),methods for modulating the activity of a polypeptide comprising analteration described herein and detection of a polypeptide comprising analteration described herein.

In another embodiment, the polypeptide or fragment is a peptide, e.g.,an immunogenic peptide or protein that contains an alteration describedherein. Such immunogenic peptides or proteins can be used to raiseantibodies specific to the polypeptide or protein comprising analteration described herein. In other embodiments, such immunogenicpeptides or proteins can be used for vaccine preparation. The vaccinepreparation can include other components, e.g., an adjuvant.

In another aspect, the invention features antibody molecules that bindto a polypeptide comprising an alteration described herein or fragmentdescribed herein. In embodiments the antibody can distinguish wild typefrom the mutated polypeptide, e.g., the polypeptide comprising analteration described herein. Techniques for generating antibodymolecules are known in the art, and are described, for example, in WO2012/092426, entitled “Optimization of Multigene Analysis of TumorSamples, incorporated herein by reference.

Detection Reagents

In another aspect, the invention features a detection reagent, e.g., apurified or an isolated preparation thereof. Detection reagents candistinguish a nucleic acid, or protein sequence, having an alterationdescribed herein, e.g., of a nucleic acid molecule comprising analteration described herein, e.g., an alteration in ERBB2; an alterationin HER2, e.g. a mutation in HER2 at residue 5310 in which residue 5310is mutated to S310F or S310Y.

Detection reagents, e.g., nucleic acid-based detection reagents, can beused to identify mutations in a target nucleic acid, e.g., DNA, e.g.,genomic DNA or cDNA, or RNA, e.g., in a sample, e.g., a sample ofnucleic acid derived from a urothelial and/or micropapillary carcinoma,e.g., MPUC. Detection reagents, e.g., antibody-based detection reagents,can be used to identify mutations in a target protein, e.g., in asample, e.g., a sample of protein derived from, or produced by, aurothelial and/or micropapillary carcinoma cell, e.g., MPUC cell.

Nucleic Acid-Based Detection Reagents

In one embodiment, the detection reagent comprises a nucleic acidmolecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, comprisingsequence which is complementary with a nucleic acid sequence on a targetnucleic acid (the sequence on the target nucleic acid that is bound bythe detection reagent is referred to herein as the “detection reagentbinding site” and the portion of the detection reagent that correspondsto the detection reagent binding site is referred to as the “targetbinding site”). In one embodiment, the detection reagent binding site isdisposed in relationship to the interrogation position such that binding(or in embodiments, lack of binding) of the detection reagent to thedetection reagent binding site allows differentiation of mutant andreference sequences for a mutant described herein (nucleic acid moleculecomprising an alteration described herein, e.g., an alteration in ERBB2;an alteration in HER2, e.g. a mutation in HER2 at residue S310 in whichresidue S310 is mutated to S310F or S310Y, or a mutation at residue 157of HER2, from a reference sequence. The detection reagent can bemodified, e.g., with a label or other moiety, e.g., a moiety that allowscapture.

In one embodiment, the detection reagent comprises a nucleic acidmolecule, e.g., a DNA, RNA or mixed DNA/RNA molecule, which, e.g., inits target binding site, includes the interrogation position and whichcan distinguish (e.g., by affinity of binding of the detection reagentto a target nucleic acid or the ability for a reaction, e.g., a ligationor extension reaction with the detection reagent) between a mutation,e.g., a translocation described herein, and a reference sequence. Inembodiments, the interrogation position can correspond to a terminal,e.g., to a 3′ or 5′ terminal nucleotide, a nucleotide immediatelyadjacent to a 3′ or 5′ terminal nucleotide, or to another internalnucleotide, of the detection reagent or target binding site.

In embodiments, the difference in the affinity of the detection reagentfor a target nucleic acid comprising the alteration described herein andthat for a target nucleic acid comprising the reference sequence allowsdetermination of the presence or absence of the mutation (or reference)sequence. Typically, such detection reagents, under assay conditions,will exhibit substantially higher levels of binding only to the mutantor only to the reference sequence, e.g., will exhibit substantial levelsof binding only to the mutation or only to the reference sequence.

In embodiments, binding allows (or inhibits) a subsequent reaction,e.g., a subsequent reaction involving the detection reagent or thetarget nucleic acid. E.g., binding can allow ligation, or the additionof one or more nucleotides to a nucleic acid, e.g., the detectionreagent, e.g., by DNA polymerase, which can be detected and used todistinguish mutant from reference. In embodiments, the interrogationposition is located at the terminus, or sufficiently close to theterminus, of the detection reagent or its target binding site, such thathybridization, or a chemical reaction, e.g., the addition of one or morenucleotides to the detection reagent, e.g., by DNA polymerase, onlyoccurs, or occurs at a substantially higher rate, when there is aperfect match between the detection reagent and the target nucleic acidat the interrogation position or at a nucleotide position within 1, 2,or 3 nucleotides of the interrogation position.

In one embodiment, the detection reagent comprises a nucleic acid, e.g.,a DNA, RNA or mixed DNA/RNA molecule wherein the molecule, or its targetbinding site, is adjacent (or flanks), e.g., directly adjacent, to theinterrogation position, and which can distinguish between a mutationdescribed herein, and a reference sequence, in a target nucleic acid.

In embodiments, the detection reagent binding site is adjacent to theinterrogation position, e.g., the 5′ or 3′terminal nucleotide of thedetection reagent, or its target binding site, is adjacent, e.g.,between 0 (directly adjacent) and 1,000, 500, 400, 200, 100, 50, 10, 5,4, 3, 2, or 1 nucleotides from the interrogation position. Inembodiments, the outcome of a reaction will vary with the identity ofthe nucleotide at the interrogation position allowing one to distinguishbetween mutant and reference sequences. E.g., in the presence of a firstnucleotide at the interrogation position a first reaction will befavored over a second reaction. E.g., in a ligation or primer extensionreaction, the product will differ, e.g., in charge, sequence, size, orsusceptibility to a further reaction (e.g., restriction cleavage)depending on the identity of the nucleotide at the interrogationposition. In embodiments the detection reagent comprises pairedmolecules (e.g., forward and reverse primers), allowing foramplification, e.g., by PCR amplification, of a duplex containing theinterrogation position. In such embodiments, the presence of themutation can be determined by a difference in the property of theamplification product, e.g., size, sequence, charge, or susceptibilityto a reaction, resulting from a sequence comprising the interrogationposition and a corresponding sequence having a reference nucleotide atthe interrogation positions. In embodiments, the presence or absence ofa characteristic amplification product is indicative of the identity ofthe nucleotide at the interrogation site and thus allows detection ofthe mutation.

In embodiments, the detection reagent, or its target binding site, isdirectly adjacent to the interrogation position, e.g., the 5′ or3′terminal nucleotide of the detection reagent is directly adjacent tothe interrogation position. In embodiments, the identity of thenucleotide at the interrogation position will determine the nature of areaction, e.g., a reaction involving the detection reagent, e.g., themodification of one end of the detection reagent. E.g., in the presenceof a first nucleotide at the interrogation position a first reactionwill be favored over a second reaction. By way of example, the presenceof a first nucleotide at the interrogation position, e.g., a nucleotideassociated with a mutation, can promote a first reaction, e.g., theaddition of a complementary nucleotide to the detection reagent. By wayof example, the presence of an A at the interrogation position willcause the incorporation of a T, having, e.g., a first colorimetriclabel, while the presence of a G and the interrogation position willcause the incorporation for a C, having, e.g., a second colorimetriclabel. In one embodiment, the presence of a first nucleotide at thenucleotide will result in ligation of the detection reagent to a secondnucleic acid. E.g., a third nucleic acid can be hybridized to the targetnucleic acid sufficiently close to the interrogation site that if thethird nucleic acid has an exact match at the interrogation site it willbe ligated to the detection reagent. Detection of the ligation product,or its absence, is indicative of the identity of the nucleotide at theinterrogation site and thus allows detection of the mutation.

A variety of readouts can be employed. E.g., binding of the detectionreagent to the mutant or reference sequence can be followed by a moiety,e.g., a label, associated with the detection reagent, e.g., aradioactive or enzymatic label. In embodiments the label comprises aquenching agent and a signaling agent and hybridization results inaltering the distance between those two elements, e.g., increasing thedistance and un-quenching the signaling agent. In embodiments, thedetection reagent can include a moiety that allows separation from othercomponents of a reaction mixture. In embodiments, binding allowscleavage of the bound detection reagent, e.g., by an enzyme, e.g., bythe nuclease activity of the DNA polymerase or by a restriction enzyme.The cleavage can be detected by the appearance or disappearance of anucleic acid or by the separation of a quenching agent and a signalingagent associated with the detection reagent. In embodiments, bindingprotects, or renders the target susceptible, to further chemicalreaction, e.g., labeling or degradation, e.g., by restriction enzymes.In embodiments binding with the detection reagent allows captureseparation or physical manipulation of the target nucleic acid tothereby allow for identification. In embodiments binding can result in adetectable localization of the detection reagent or target, e.g.,binding could capture the target nucleic acid or displace a thirdnucleic acid. Binding can allow for the extension or other size changein a component, e.g., the detection reagent, allowing distinctionbetween mutant and reference sequences. Binding can allow for theproduction, e.g., by PCR, of an amplicon that distinguishes mutant fromreference sequence.

In one embodiment the detection reagent, or the target binding site, isbetween 5 and 500, 5 and 300, 5 and 250, 5 and 200, 5 and 150, 5 and100, 5 and 50, 5 and 25, 5 and 20, 5 and 15, or 5 and 10 nucleotides inlength. In one embodiment the detection reagent, or the target bindingsite, is between 10 and 500, 10 and 300, 10 and 250, 10 and 200, 10 and150, 10 and 100, 10 and 50, 10 and 25, 10 and 20, or 10 and 15,nucleotides in length. In one embodiment the detection reagent, or thetarget binding site, is between 20 and 500, 20 and 300, 20 and 250, 20and 200, 20 and 150, 20 and 100, 20 and 50, or 20 and 25 nucleotides inlength. In one embodiment the detection reagent, or the target bindingsite, is sufficiently long to distinguish between mutant and referencesequences and is less than 100, 200, 300, 400, or 500 nucleotides inlength.

Preparations of Nucleic Acids and Uses Thereof

In another aspect, the invention features purified or isolatedpreparations of a neoplastic or tumor cell nucleic acid, e.g., DNA,e.g., genomic DNA or cDNA, or RNA, containing an interrogation positiondescribed herein, useful for determining if a mutation disclosed hereinis present. The nucleic acid includes the interrogation position, andtypically additional sequence on one or both sides of the interrogationposition. In addition the nucleic acid can contain heterologoussequences, e.g., adaptor or priming sequences, typically attached to oneor both terminus of the nucleic acid. The nucleic acid also includes alabel or other moiety, e.g., a moiety that allows separation orlocalization.

In embodiments, the nucleic acid is between 20 and 1,000, 30 and 900, 40and 800, 50 and 700, 60 and 600, 70 and 500, 80 and 400, 90 and 300, or100 and 200 nucleotides in length (with or without heterologoussequences). In one embodiment, the nucleic acid is between 40 and 1,000,50 and 900, 60 and 800, 70 and 700, 80 and 600, 90 and 500, 100 and 400,110 and 300, or 120 and 200 nucleotides in length (with or withoutheterologous sequences). In another embodiment, the nucleic acid isbetween 50 and 1,000, 50 and 900, 50 and 800, 50 and 700, 50 and 600, 50and 500, 50 and 400, 50 and 300, or 50 and 200 nucleotides in length(with or without heterologous sequences). In embodiments, the nucleicacid is of sufficient length to allow sequencing (e.g., by chemicalsequencing or by determining a difference in T_(m) between mutant andreference preparations) but is optionally less than 100, 200, 300, 400,or 500 nucleotides in length (with or without heterologous sequences).

Such preparations can be used to sequence nucleic acid from a sample,e.g., a neoplastic or tumor sample. In one embodiment the purifiedpreparation is provided by in situ amplification of a nucleic acidprovided on a substrate. In embodiments the purified preparation isspatially distinct from other nucleic acids, e.g., other amplifiednucleic acids, on a substrate.

In one embodiment, the purified or isolated preparation of nucleic acidis derived from a urothelial and/or micropapillary carcinoma, e.g.,MPUC.

Such preparations can be used to determine if a sample comprises mutantsequence, e.g., an alteration described herein.

In another aspect, the invention features, a method of determining thesequence of an interrogation position for an alteration describedherein, comprising:

providing a purified or isolated preparations of nucleic acid, e.g.,DNA, e.g., genomic DNA or cDNA, or RNA, containing an interrogationposition described herein,

sequencing, by a method that breaks or forms a chemical bond, e.g., acovalent or non-covalent chemical bond, e.g., in a detection reagent ora target sequence, the nucleic acid so as to determine the identity ofthe nucleotide at an interrogation position. The method allowsdetermining if an alteration described herein is present.

In one embodiment, sequencing comprises contacting the nucleic acidcomprising an alteration described herein with a detection reagentdescribed herein.

In one embodiment, sequencing comprises determining a physical property,e.g., stability of a duplex form of the nucleic acid comprising analteration described herein, e.g., T_(m), that can distinguish mutantfrom reference sequence.

In one embodiment, the nucleic acid comprising an alteration describedherein is derived from a urothelial and/or micropapillary carcinoma,e.g., MPUC.

Reaction Mixtures and Devices

In another aspect, the invention features, purified or isolatedpreparations of a nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, orRNA, containing an interrogation position described herein, useful fordetermining if a mutation disclosed herein is present, disposed insequencing device, or a sample holder for use in such a device. In oneembodiment, the nucleic acid is derived from a urothelial and/ormicropapillary carcinoma, e.g., MPUC.

In another aspect, the invention features, purified or isolatedpreparations of a nucleic acid, e.g., DNA, e.g., genomic DNA or cDNA, orRNA, containing an interrogation position described herein, useful fordetermining if a mutation disclosed herein is present, disposed in adevice for determining a physical or chemical property, e.g., stabilityof a duplex, e.g., T_(m)or a sample holder for use in such a device. Inone embodiment, the device is a calorimeter. In one embodiment thenucleic acid comprising an alteration described herein is derived from aurothelial and/or micropapillary carcinoma, e.g., MPUC.

The detection reagents described herein can be used to determine if analteration described herein is present in a sample. In embodiments, thesample comprises a nucleic acid that is derived from a micropapillarycarcinoma, e.g., MPUC. The cell can be from a neoplastic or a tumorsample, e.g., a biopsy taken from the neoplasm or the tumor; fromcirculating tumor cells, e.g., from peripheral blood; or from a blood orplasma sample. In one embodiment, the nucleic acid is derived from aurothelial and/or micropapillary carcinoma, e.g., MPUC.

Accordingly, in one aspect, the invention features a method of making areaction mixture, comprising:

combining a detection reagent, or purified or isolated preparationthereof, described herein with a target nucleic acid derived from aurothelial and/or micropapillary carcinoma, e.g., MPUC, which comprisesa sequence having an interrogation position for an alteration describedherein.

In another aspect, the invention features a reaction mixture,comprising:

a detection reagent, or purified or isolated preparation thereof,described herein; and

a target nucleic acid derived from a urothelial and/or micropapillarycarcinoma cell, e.g., a MPUC cell, which comprises a sequence having aninterrogation position for an alteration described herein.

In one embodiment of the reaction mixture, or the method of making thereaction mixture:

the detection reagent comprises a nucleic acid, e.g., a DNA, RNA ormixed DNA/RNA, molecule which is complementary with a nucleic acidsequence on a target nucleic acid (the detection reagent binding site)wherein the detection reagent binding site is disposed in relationshipto the interrogation position such that binding of the detection reagentto the detection reagent binding site allows differentiation of mutantand reference sequences for a mutation sequence or event describedherein.

In one embodiment of the reaction mixture, or the method of making thereaction mixture: the target nucleic acid sequence is derived from aurothelial and/or micropapillary carcinoma, e.g., MPUC, as describedherein. In one embodiment of the reaction mixture, or the method ofmaking the reaction mixture: the mutation is an alteration describedherein, including: a substitution, e.g., a substitution describedherein.

An alteration described herein, can be distinguished from a reference,e.g., a non-mutant or wildtype sequence, by reaction with an enzyme thatreacts differentially with the mutation and the reference. E.g., theycan be distinguished by cleavage with a restriction enzyme that hasdiffering activity for the mutant and reference sequences. E.g., theinvention includes a method of contacting a nucleic acid comprising analteration described herein with such an enzyme and determining if aproduct of that cleavage which can distinguish mutant form referencesequence is present.

In one aspect the inventions provides, a purified preparation of arestriction enzyme cleavage product which can distinguish between mutantand reference sequence, wherein one end of the cleavage product isdefined by an enzyme that cleaves differentially between mutant andreference sequence. In one embodiment, the cleavage product includes theinterrogation position.

Protein-Based Detection Reagents, Methods, Reaction Mixtures and Devices

A mutant protein described herein can be distinguished from a reference,e.g., a non-mutant or wild-type protein, by reaction with a reagent,e.g., a substrate, e.g, a substrate for catalytic activity or functionalactivity, or an antibody, that reacts differentially with the mutant andreference protein. In one aspect, the invention includes a method ofcontacting a sample comprising a mutant protein described herein withsuch reagent and determining if the mutant protein is present in thesample.

In another embodiment, the invention features, an antibody that candistinguish a mutant protein described herein, or a fragment thereof,from a reference, e.g., a non-mutant or wild type protein.

Accordingly, in one aspect, the invention features a method of making areaction mixture comprising:

combining a detection reagent, or purified or isolated preparationthereof, e.g., a substrate, e.g., a substrate for phosphorylation orother activity, or an antibody, described herein with a target proteinderived from a urothelial and/or micropapillary carcinoma cell, e.g.,MPUC cell, which comprises a sequence having an interrogation positionfor an alteration described herein.

In another aspect, the invention features, a reaction mixturecomprising:

a detection reagent, or purified or isolated preparation thereof, e.g.,a substrate, e.g., a substrate for phosphorylation or other activity, oran antibody, described herein; and

a target protein derived from a urothelial and/or micropapillarycarcinoma cell, e.g., MPUC cell, which comprises a sequence having aninterrogation position for an alteration described herein.

In one embodiment of the reaction mixture, or the method of making thereaction mixture:

the detection reagent comprises an antibody specific for a mutantprotein described herein.

In one embodiment of the reaction mixture, or the method of making thereaction mixture that includes a urothelial and/or micropapillarycarcinoma cell, e.g., MPUC cell.

In one embodiment of the reaction mixture, or the method of making thereaction mixture: the mutation is an alteration described herein (e.g.,a HER2 mutation described herein).

Screening Methods

In another aspect, the invention features a method, or assay, forscreening for agents that modulate, e.g., inhibit, the expression oractivity of a nucleic acid or polypeptide or protein comprising amutation as described herein. The method includes contacting a nucleicacid or polypeptide or protein comprising an alteration describedherein, or a cell expressing a nucleic acid or polypeptide or proteincomprising an alteration described herein, with a candidate agent; anddetecting a change in a parameter associated with a nucleic acid orpolypeptide or protein comprising an alteration described herein, e.g.,a change in the expression or an activity of the nucleic acid orpolypeptide or protein comprising an alteration described herein. Themethod can, optionally, include comparing the treated parameter to areference value, e.g., a control sample (e.g., comparing a parameterobtained from a sample with the candidate agent to a parameter obtainedfrom a sample without the candidate agent). In one embodiment, if adecrease in expression or activity of the nucleic acid or polypeptide orprotein comprising an alteration described herein is detected, thecandidate agent is identified as an inhibitor. In another embodiment, ifan increase in expression or activity of the nucleic acid or polypeptideor protein comprising an alteration described herein is detected, thecandidate agent is identified as an activator.

In one embodiment, the contacting step is effected in a cell-freesystem, e.g., a cell lysate or in a reconstituted system. In otherembodiments, the contacting step is effected in a cell in culture, e.g.,a cell expressing an alteration described herein (e.g., a mammaliancell, a tumor cell or cell line, a recombinant cell). In yet otherembodiments, the contacting step is effected in a cell in vivo(a—expressing cell present in a subject, e.g., an animal subject (e.g.,an in vivo animal model).

Exemplary parameters evaluated include one or more of:

(i) a change in binding activity, e.g., direct binding of the candidateagent to a polypeptide comprising an alteration described herein; abinding competition between a known ligand and the candidate agent to apolypeptide comprising an alteration described herein;

(ii) a change in kinase activity, e.g., phosphorylation levels of apolypeptide comprising an alteration described herein (e.g., anincreased or decreased autophosphorylation); or a change in a target ofa polypeptide comprising an alteration described herein, In certainembodiments, a change in kinase activity, e.g., phosphorylation, isdetected by any of Western blot (e.g., using an antibody which binds toa polypeptide comprising an alteration described herein, massspectrometry, immunoprecipitation, immunohistochemistry, immunomagneticbeads, among others;

(iii) a change in an activity of a cell containing a tumor cell or arecombinant cell, e.g., a change in proliferation, morphology ortumorigenicity of the cell;

(iv) a change in tumor present in an animal subject, e.g., size,appearance, proliferation, of the tumor; or

(v) a change in the level, e.g., expression level, of a nucleic acid orpolypeptide or protein comprising an alteration described herein.

In one embodiment, a change in a cell free assay in the presence of acandidate agent is evaluated. For example, an activity of a nucleic acidor polypeptide or protein comprising an alteration described herein, orinteraction of a nucleic acid or polypeptide or protein comprising analteration described herein with a downstream ligand can be detected. Inone embodiment, the polypeptide or protein comprising an alterationdescribed herein is contacted with a ligand, e.g., in solution, and acandidate agent is monitored for an ability to modulate, e.g., inhibit,an interaction, e.g., binding, between the nucleic acid or polypeptideor protein comprising an alteration described herein and the ligand.

In other embodiments, a change in an activity of a cell is detected in acell in culture, e.g., a cell expressing a mutation (e.g., a mammaliancell, a tumor cell or cell line, a recombinant cell). In one embodiment,the cell is a recombinant cell that is modified to express a nucleicacid comprising an alteration described herein, e.g., is a recombinantcell transfected with a nucleic acid comprising an alteration describedherein. The transfected cell can show a change in response to theexpressed mutation, e.g., increased proliferation, changes inmorphology, increased tumorigenicity, and/or acquired a transformedphenotype. A change in any of the activities of the cell, e.g., therecombinant cell, in the presence of the candidate agent can bedetected. For example, a decrease in one or more of: proliferation,tumorigenicity, transformed morphology, in the presence of the candidateagent can be indicative of an inhibitor of a nucleic acid or polypeptideor protein comprising an alteration described herein. In otherembodiments, a change in binding activity or phosphorylation asdescribed herein is detected.

In yet other embodiment, a change in a tumor present in an animalsubject (e.g., an in vivo animal model) is detected. In one embodiment,the animal model is a tumor containing animal or a xenograft comprisingcells expressing a nucleic acid or polypeptide or protein comprising analteration described herein (e.g., tumorigenic cells expressing anucleic acid or polypeptide or protein comprising an alterationdescribed herein). The candidate agent can be administered to the animalsubject and a change in the tumor is detected. In one embodiment, thechange in the tumor includes one or more of a tumor growth, tumor size,tumor burden, survival, is evaluated. A decrease in one or more of tumorgrowth, tumor size, tumor burden, or an increased survival is indicativethat the candidate agent is an inhibitor.

In other embodiments, a change in expression of a nucleic acid orpolypeptide or protein comprising an alteration described herein can bemonitored by detecting the nucleic acid or protein levels, e.g., usingthe methods described herein.

In certain embodiments, the screening methods described herein can berepeated and/or combined. In one embodiment, a candidate agent that isevaluated in a cell-free or cell-based described herein can be furthertested in an animal subject.

In one embodiment, the candidate agent is a small molecule compound,e.g., a kinase inhibitor, a nucleic acid (e.g., antisense, siRNA,aptamer, ribozymes, microRNA), an antibody molecule (e.g., a fullantibody or antigen binding fragment thereof that binds to themutation). The candidate agent can be obtained from a library (e.g., acommercial library of kinase inhibitors) or rationally designed.

In other embodiments, the method, or assay, includes providing a stepbased on proximity-dependent signal generation, e.g., a two-hybrid assaythat includes a first mutation protein (e.g., a mutated protein), and asecond mutated protein (e.g., a ligand), contacting the two-hybrid assaywith a test compound, under conditions wherein said two hybrid assaydetects a change in the formation and/or stability of the complex, e.g.,the formation of the complex initiates transcription activation of areporter gene.

In one non-limiting example, the three-dimensional structure of theactive site of a polypeptide or protein comprising an alterationdescribed herein is determined by crystallizing the complex formed bythe polypeptide or protein and a known inhibitor. Rational drug designis then used to identify new test agents by making alterations in thestructure of a known inhibitor or by designing small molecule compoundsthat bind to the active site of the polypeptide or protein.

The candidate agents can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; peptoid libraries (libraries of molecules havingthe functionalities of peptides, but with a novel, non-peptide backbonewhich are resistant to enzymatic degradation but which neverthelessremain bioactive; see, e.g., Zuckermann, R. N. et al. (1994) J. Med.Chem. 37:2678-85); spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are limited to peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner,U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. 5,223,409),plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or onphage (Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382;Felici (1991) J. Mol. Biol. 222:301-310; Ladner supra.).

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). An FET binding event can be conveniently measured throughstandard fluorometric detection means known in the art (e.g., using afluorimeter).

In another embodiment, determining the ability of the mutated protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. andUrbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995)Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or“BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

EXAMPLES Example 1. An Activating Extracellular Domain ERBB2 (HER2)Mutation in Urothelial Carcinoma (UC)

This example describes the results from a study of 35 UC which includeda subset of cases used in Example 2. Experimental conditions are asdescribed in Example 2. A single ERBB2 mutation in the only MPUC caseprofiled is described.

In this study, the UC in a 71-year-old female patient with stage IV highgrade UC, an S310F external domain mutation in the ERBB2 gene wasidentified. This is the first reported mutation of ERBB2 in UC. Thehistology is taken from the lymph node metastasis specimen used for theNGS assessment. Note that this tumor has a micropapillary architecture.This UC features the S310F base substitution in the ERBB2 (HER2) gene.This tumor also featured mutations in the FBXW7 and TP53 genes.

Recent in vitro data suggest that ERBB2 S310F is an activating mutation,which is sensitive to irreversible dual Egfr/Erbb2 inhibitors (GreulichH (2010) Cancer Res. 1:1200-1210). ERBB2 mutations have not beenpreviously reported in urothelial carcinoma (COSMIC, PubMed, August2012), yet suggesting sensitivity to Her2-targeted drug therapies.

Example 2. A High Frequency of Activating Extracellular Domain ERBB2(HER2) Mutation in Micropapillary Urothelial Carcinoma

In this example, a genomic analysis was conducted of a series ofpatients with MPUC and an expanded series of non-MPUC to characterizethe genomic landscape of MUPC and to identify targeted treatment optionsfor patients diagnosed with this disease.

Methods

Targeted next generation sequencing (NGS) was performed onhybridization-captured, adaptor ligation based libraries using DNAextracted from 4 formalin-fixed paraffin embedded sections cut at 10microns from 15 cases of MPUC and 64 cases of non-MUPC in aCLIA-certified lab (Foundation Medicine). The pathologic diagnosis ofeach case was confirmed on routine hematoxylin and eosin stained slidesand all samples forwarded for DNA extraction contained a minimum of 20%DNA derived from tumor cells. All MPUC were histologically confirmedused published criteria (Sangoi A R, et al. (2010) Am J Surg Pathol.34:1367-76) by 3 pathologists. DNA sequencing was performed for 3,230exons of 182 cancer-related genes and 37 introns of 14 genes frequentlyrearranged in cancer (1.14 million total bps) on indexed, adaptorligated, hybridization-captured (Agilent SureSelect custom kit) andfully sequenced using 49 bp paired reads on the Illumina HiSeq 2000. TheMPUC cases were sequenced to at an average depth of 978X. The non-MP UCwere sequenced to an average depth of 969X. All samples were evaluatedfor genomic alterations including base substitutions, insertions,deletions, copy number alterations (amplifications and homozygousdeletions), and select gene fusions/rearrangements as previouslydescribed (Lipson D, et al. (2012) Nat Med. 18:382-4).

The bioinformatics processes used in this study included Bayesianalgorithms to detect base substitutions, local assembly algorithms todetect short insertions and deletions, a comparison with process-matchednormal control samples to detect gene copy number alterations and ananalysis of chimeric read pairs to identify gene fusions. Actionable GAwere defined as being linked to commercially available targetedtherapies on the market or to targeted therapies being tested registeredclinical trials. The 10 MPUC cases tested for HER2 (ERBB2) proteinoverexpression by immunohistochemistry (IHC) using the Dako Herceptestassay.

Results

The 15 MPUC samples were obtained from 10 male (66%) and 5 female (33%)patients with a mean age of 66 years (range 55 to 86 years). Sequencingwas performed on the primary tumor in 11 (73%) cases (6 TURBT samplesand 5 cystectomies) and on metastatic lesions in 4 (27%) of cases. Alltumors were high grade, 3 cases were stage I, 3 were stage II, 2 werestage III and 7 were stage IV. A total of 67 genomic alterations (GA)(average 4.47 GA per tumor) were identified including alterations inTP53 (10 cases, 67%), ERBB2 (6 cases, 40%), MCL1 (5 cases, 33%), RB1 (5cases, 33%), and ARID1A (4 cases, 27%) (FIG. 1 and FIG. 2). The 6 ERBB2mutations were all located within the extracellular domain of ERBB2including S310F (4 cases), S310Y (1 case) and R157W (1 case) (FIG. 2;FIG. 5). No mutations in the ERBB2 tyrosine kinase domain were observed.All 6 cases of MPUC with ERBB2 mutation were negative for ERBB2amplification and in the 3 cases where additional tissue was availablefor testing, these ERBB2 mutated MPUC were also negative for HER2over-expression by immunohistochemistry (IHC) (FIG. 2; FIG. 5).

In contrast, only 6/64 (9.4%) of non-MP UC harbored ERBB2 alterationsincluding S310F mutations (3 cases), amplification (2 cases, 40 and 15copies) and an ERBB2-GRB7 fusion (1 case) (FIG. 4; FIG. 6). The ERBB2mutation frequency observed in both the MPUC and non-MPUC cohorts arehigher than the 2/159 (1.3%) of protein changing ERBB2 mutationsreported in urinary tract cancer in COSMIC (15). The enrichment of ERBB2alterations in MPUC compared to non-MP UC is significant between thisseries (p<0.0084) and for all types of urinary tract cancer in COSMIC(p<0.001). All 9 ERBB2 WT MPUC cases harbored at least 1 actionablealteration, including alterations in AKT1, AKT2, CCND1, EGFR, PIK3CA,PIK3R1 and RAF1. The most frequent alterations in the non-MPUC groupinvolved mutations in TP53 (38 total; 58% of non-MPUC cases) andCDKN2A/B (25 total; 39% of non-MPUC cases). Alterations in chromatinremodeling genes including truncating mutations in KDM6A (17 total; 27%of non-MPUC cases) and ARID1A (12 total; 19% of non-MPUC cases) werenotable in the non-MPUC group (FIG. 4; FIG. 6).

Discussion

MPUC is a relatively rare subtype of UC which comprises approximately3,000 to 4,000 new cases diagnosed each year in the US, a an incidencejust below that of the successfully targeted disease, chronicmyelogenous leukemia (Amin M B, et al. (1994) Am J Surg Pathol.18:1224-32); Sangoi A R, et al. (2010) Am J Surg Pathol. 34:1367-76;Kamat A M, et al. (2007) Cancer. 110:62-7; López J I, et al. (1999)Histopathology 34:561-2). MUPC is widely considered to have an adverseprognosis reflected in the propensity to invade lymphovascular spacesand spread to distant sites early in the course of the disease (Amin MB, et al. (1994) supra); Sangoi A R, et al. (2010) supra; Kamat A M, etal. (2007); López J I, et al. (1999) supra). Any component of MPUC in aUC of the bladder is considered to be significant and studies have shownthat, as the proportion of the MPUC component increases, the prognosisworsens (Amin, M D. (2009) Modern Pathol. 22:S96-S118; Samaratunga H, etal. (2004) Histopathol. 45: 55-64; Stewart S L, et al. (2004) MMWRSurveill Summ. 53:1-108). Since MPUC is well-known to metastasize evenwhen local invasion of the bladder muscle wall is absent, early radicalsurgery has been recommended for MPUC in comparison with conventionalnon-MP UC (Kamat A M, et al. (2007) supra.

In the present example, the significant enrichment of ERBB2 mutationfrequency observed in MPUC (40%) versus non-MPUC (9.4%) was observed(p<0.0084). When compared with the COSMIC database which contains only 2(1.4%) protein altering mutations in ERBB2 in 158 carcinomas of theurinary bladder (COSMIC v65 Release”. Catalogue Of Somatic Mutations InCancer. Wellcome Trust Sanger Institute. Retrieved 1 Jul. 2013(http://www.sanger.ac.uk/cosmic)), the enrichment in MPUC is also highlysignificant (p<0.0001).

All 6ERBB2 mutations identified in MPUC in this study were localized tothe extracellular domain, with five mutations at 5310 and no mutationswithin the tyrosine kinase domain. This contrasts with other tumor typeswhere the majority of ERBB2 mutations are located within the kinasedomain with a frequency in COSMIC of 78% in all tissues, 97% in lungadenocarcinoma and 81% in breast cancer (FIG. 3). The biologicalunderpinnings of the extracellular domain location of the MUPC ERBB2mutations are unclear and warrant further investigation.

In contrast, in lung cancer ERBB2 alterations are predominantlyinsertion mutations in the kinase with only a small fraction ofalterations involving the extra-cellular domain (COSMIC v65 Release”.Catalogue Of Somatic Mutations In Cancer. Wellcome Trust SangerInstitute. Retrieved 1 Jul. 2013 (http://www.sanger.ac.uk/cosmic). Inbreast cancer, although similar to lung cancer in the localization ofthe large majority of ERBB2 alterations to the kinase domain, insertionsare relatively uncommon and virtually all of the kinase domain mutationsare base substitutions (COSMIC v65 Release”. Catalogue Of SomaticMutations In Cancer. Wellcome Trust Sanger Institute. Retrieved 1 Jul.2013 (http://www.sanger.ac.uk/cosmic). An enrichment of ERBB2 mutationwithin a common cancer subtype has also been recently described in aseries of CDH1 mutated invasive lobular carcinomas of the breast breastwith a frequency of 23% compared to a frequency of 2% in all breastcancers (Ross J. S., et al. (2013) Clin Cancer Res. 19:2668-76).

In the Bladder Urothelial Carcinoma TCGA dataset, although mutations inERBB2 are not described, amplification of ERBB2 was listed in 6% (9/150) of cases (The cBio Cancer Genomics Portal, April 2013). Separatestudies have reported ERBB2 amplification predominantly based offluorescence in situ hybridization (FISH) analysis in 8-9% of primaryurothelial carcinomas, and at a higher frequency in lymph nodemetastases (Fleischmann A, et al. (2011) Eur Urol. 60:350-7). Inaddition, in a study of non-muscle-invasive bladder cancers, ERBB2amplification has been observed in high grade urothelial carcinomas(HG-UCs) at a similar incidence of 9%, but not in any of the papillaryurothelial neoplasms of low malignant potential or low grade urothelialcarcinomas (LG-UCs) studied, and has been associated with recurrence andprogression in high grade UC (Chen P C, (2013) et al. J Clin Pathol.66:113-9). Her2 overexpression has been identified in 19% ( 22/116) ofbladder cancers, with significant enrichment in grade III and muscleinvasive tumors Gardiner R A, et al. (1992) Urol Res. 20:117-20).However, studies have reported inconsistent results regarding theprognostic value of HER2 expression detected by IHC (Tsai Y S, et al.(2012) Adv Urol.:181964). In the current study, 3 (100%) of ERBB2mutated MPUC were negative for HER2 expression by IHC.

In addition to ERBB2 amplification, activating ERBB2 mutations can alsopredict sensitivity to anti-HER2-targeted therapies (Herter-Sprie G S,et al. Front Oncol. 2013; 3:86; Bose, et al. (2013) Cancer Discov.3(2):224-37; Mazieres J, et al. (2013) J Clin Oncol. 31:1997-2003; Ali SM, et al. (2013) J Clin Oncol. September 27 [Epub ahead of print).Irreversible HER2 (ERBB2) inhibitors are emerging and appear to showgreater potency and durability than on the market reversible inhibitorsin both clinical and preclinical settings (Bose, et al. (2013) CancerDiscov. 3(2):224-37; Mazieres J, et al. (2013) J Clin Oncol.31:1997-2003). The S310F/Y ERBB2 extra-cellular domain mutations seen in5 cases of MPUC and 3 cases of UC is considered to be an activatingmutation and sensitive to irreversible dual Egfr/Erbb2 inhibitors(Herter-Sprie G S, et al. (2013) Front Oncol. 3:86; Lee J C, et al.(2006) PLoS Med. 3:e485; Greulich H. et al. Genes Cancer. 2010;1:1200-10; Greulich H, et al. (2012) Proc Natl Acad Sci USA.109:14476-81). The kinase domain alterations in ERBB2 are considered tobe homologous to those encountered in the EGFR gene (Greulich H. (2010)Genes Cancer 1:1200-10). The S310F/Y extracellular domain ERBB2mutations found in the MPUC cases are not as comparable to EGFRextra-cellular domain mutations are, to date, not as well characterizedas the insertion and base substitution mutations described in lung andbreast cancers (COSMIC v65 Release”. Catalogue Of Somatic Mutations InCancer. Wellcome Trust Sanger Institute. Retrieved 1 Jul. 2013(http://www.sanger.ac.uk/cosmic). Although the mechanism of receptoractivation has not yet been characterized for these EGFR extracellulardomain mutations, it is tempting to speculate that the underlyingtumorigenic mechanism is caused by a less tethered conformation of theextracellular domain as most amino acid substitutions localize tointerdomain interfaces (Lee J C, et al. (2006) PLoS Med 3:e485).

Although the currently available anti-HER2 targeted therapies such astrastuzumab, lapatanib, and others are currently under investigation fortreatment of ERBB2-amplified urothelial carcinomas, Phase 3 trial datahas yet to emerge (Marín A P, et al. (2010) J Cancer Res Clin Oncol.136:1915-20. For non-amplified bladder cancers such as the 6 ERBB2mutated MPUC in the current study, the standard tests for HER2 (ERBB2)amplification/overexpression status (IHC and FISH) were uniformlynegative and these aggressive tumors would thus not have been detectedas being driven by ERBB2 activation. Given the promise of targetingERBB2 mutated (HER2 IHC/FISH negative) tumors such as has recentlystarted in the clinical trials setting for breast and lung cancers, thecurrent study argues that this approach should be extended to urinarybladder cancer especially when the tumor features an MPUC pattern. Themicropapillary architecture and well-documented aggressive clinicalcourse attributed MPUC has also been linked to micropapillary carcinomasof the endometrium, breast and lung (del Carmen M G, et al. (2012)Gynecol Oncol. 127:651-61; Chen L, et al. (2008) Int J Surg Pathol.;16:155-63; Kamiya K, et al. (2008) Mod Pathol. 21:992-1001). However, todate there has been no reported association of ERBB2 mutations in theseother aggressive types of micropapillary carcinomas. This studyillustrates the impact of histologic subtyping on the genomic landscapeand the resulting potential to direct targeted therapies.

An exemplary amino acid and nucleotide sequence for human HER2 areprovided herein as SEQ ID NO:1 and SEQ ID NO:2, respectively. SEQ IDNO:1 corresponds to a HER2 isoform that contains a signal peptide (aminoacids 1-22). Residue Ser310 is highlighted.

NCBI Reference Sequence: NP_004439.2 (SEQ ID NO: 1)    1melaalcrwg lllallppga astqvctgtd mldrlpaspe thldmlrhly qgcqvvqgnl   61eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng  121dpinnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla  181ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc  241aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp  301ynylstdvg s  ctivcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan  361iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf etleeitgyl yisawpdslp  421dlsvfqnlqv irgrilhnga ysltlqglgi swlglislre lgsglalihh nthlcfvhtv  481pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec  541veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc  601psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisavvg  661illvvvlgvv fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel  721rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp  781yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr  841lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft  901hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm  961idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl dstfyrslle dddmgdlvda 1021eeylvpqqgf fcpdpapgag gmvhhrhrss strsgggdlt lglepseeea prsplapseg 1081agsdvfdgdl gmgaakglqs lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv 1141nqpdvrpqpp spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq 1201ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg ldvpvNCBI Reference Sequence: NM_004448.2 (SEQ ID NO: 2)    1atggagctgg cggccttgtg ccgctggggg ctcctcctcg ccctcttgcc ccccggagcc   61gcgagcaccc aagtgtgcac cggcacagac atgaagctgc ggctccctgc cagtcccgag  121acccacctgg acatgctccg ccacctctac cagggctgcc aggtggtgca gggaaacctg  181gaactcacct acctgcccac caatgccagc ctgtccttcc tgcaggatat ccaggaggtg  241cagggctacg tgctcatcgc tcacaaccaa gtgaggcagg tcccactgca gaggctgcgg  301attgtgcgag gcacccagct ctttgaggac aactatgccc tggccgtgct agacaatgga  361gacccgctga acaataccac ccctgtcaca ggggcctccc caggaggcct gcgggagctg  421cagcttcgaa gcctcacaga gatcttgaaa ggaggggtct tgatccagcg gaacccccag  481ctctgctacc aggacacgat tttgtggaag gacatcttcc acaagaacaa ccagctggct  541ctcacactga tagacaccaa ccgctctcgg gcctgccacc cctgttctcc gatgtgtaag  601ggctcccgct gctggggaga gagttctgag gattgtcaga gcctgacgcg cactgtctgt  661gccggtggct gtgcccgctg caaggggcca ctgcccactg actgctgcca tgagcagtgt  721gctgccggct gcacgggccc caagcactct gactgcctgg cctgcctcca cttcaaccac  781agtggcatct gtgagctgca ctgcccagcc ctggtcacct acaacacaga cacgtttgag  841tccatgccca atcccgaggg ccggtataca ttcggcgcca gctgtgtgac tgcctgtccc  901tacaactacc tttctacgga cgtgggatcc tgcaccctcg tctgccccct gcacaaccaa  961gaggtgacag cagaggatgg aacacagcgg tgtgagaagt gcagcaagcc ctgtgcccga 1021gtgtgctatg gtctgggcat ggagcacttg cgagaggtga gggcagttac cagtgccaat 1081atccaggagt ttgctggctg caagaagatc tttgggagcc tggcatttct gccggagagc 1141tttgatgggg acccagcctc caacactgcc ccgctccagc cagagcagct ccaagtgttt 1201gagactctgg aagagatcac aggttaccta tacatctcag catggccgga cagcctgcct 1261gacctcagcg tcttccagaa cctgcaagta atccggggac gaattctgca caatggcgcc 1321tactcgctga ccctgcaagg gctgggcatc agctggctgg ggctgcgctc actgagggaa 1381ctgggcagtg gactggccct catccaccat aacacccacc tctgcttcgt gcacacggtg 1441ccctgggacc agctctttcg gaacccgcac caagctctgc tccacactgc caaccggcca 1501gaggacgagt gtgtgggcga gggcctggcc tgccaccagc tgtgcgcccg agggcactgc 1561tggggtccag ggcccaccca gtgtgtcaac tgcagccagt tccttcgggg ccaggagtgc 1621gtggaggaat gccgagtact gcaggggctc cccagggagt atgtgaatgc caggcactgt 1681ttgccgtgcc accctgagtg tcagccccag aatggctcag tgacctgttt tggaccggag 1741gctgaccagt gtgtggcctg tgcccactat aaggaccctc ccttctgcgt ggcccgctgc 1801cccagcggtg tgaaacctga cctctcctac atgcccatct ggaagtttcc agatgaggag 1861ggcgcatgcc agccttgccc catcaactgc acccactcct gtgtggacct ggatgacaag 1921ggctgccccg ccgagcagag agccagccct ctgacgtcca tcatctctgc ggtggttggc 1981attctgctgg tcgtggtctt gggggtggtc tttgggatcc tcatcaagcg acggcagcag 2041aagatccgga agtacacgat gcggagactg ctgcaggaaa cggagctggt ggagccgctg 2101acacctagcg gagcgatgcc caaccaggcg cagatgcgga tcctgaaaga gacggagctg 2161aggaaggtga aggtgcttgg atctggcgct tttggcacag tctacaaggg catctggatc 2221cctgatgggg agaatgtgaa aattccagtg gccatcaaag tgttgaggga aaacacatcc 2281cccaaagcca acaaagaaat cttagacgaa gcatacgtga tggctggtgt gggctcccca 2341tatgtctccc gccttctggg catctgcctg acatccacgg tgcagctggt gacacagctt 2401atgccctatg gctgcctctt agaccatgtc cgggaaaacc gcggacgcct gggctcccag 2461gacctgctga actggtgtat gcagattgcc aaggggatga gctacctgga ggatgtgcgg 2521ctcgtacaca gggacttggc cgctcggaac gtgctggtca agagtcccaa ccatgtcaaa 2581attacagact tcgggctggc tcggctgctg gacattgacg agacagagta ccatgcagat 2641gggggcaagg tgcccatcaa gtggatggcg ctggagtcca ttctccgccg gcggttcacc 2701caccagagtg atgtgtggag ttatggtgtg actgtgtggg agctgatgac ttttggggcc 2761aaaccttacg atgggatccc agcccgggag atccctgacc tgctggaaaa gggggagcgg 2821ctgccccagc cccccatctg caccattgat gtctacatga tcatggtcaa atgttggatg 2881attgactctg aatgtcggcc aagattccgg gagttggtgt ctgaattctc ccgcatggcc 2941agggaccccc agcgctttgt ggtcatccag aatgaggact tgggcccagc cagtcccttg 3001gacagcacct tctaccgctc actgctggag gacgatgaca tgggggacct ggtggatgct 3061gaggagtatc tggtacccca gcagggcttc ttctgtccag accctgcccc gggcgctggg 3121ggcatggtcc accacaggca ccgcagctca tctaccagga gtggcggtgg ggacctgaca 3181ctagggctgg agccctctga agaggaggcc cccaggtctc cactggcacc ctccgaaggg 3241gctggctccg atgtatttga tggtgacctg ggaatggggg cagccaaggg gctgcaaagc 3301ctccccacac atgaccccag ccctctacag cggtacagtg aggaccccac agtacccctg 3361ccctctgaga ctgatggcta cgttgccccc ctgacctgca gcccccagcc tgaatatgtg 3421aaccagccag atgttcggcc ccagccccct tcgccccgag agggccctct gcctgctgcc 3481cgacctgctg gtgccactct ggaaaggccc aagactctct ccccagggaa gaatggggtc 3541gtcaaagacg tttttgcctt tgggggtgcc gtggagaacc ccgagtactt gacaccccag 3601ggaggagctg cccctcagcc ccaccctcct cctgccttca gcccagcctt cgacaacctc 3661tattactggg accaggaccc accagagcgg ggggctccac ccagcacctt caaagggaca 3721cctacggcag agaacccaga gtacctgggt ctggacgtgc cagtgtga

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed by the following claims.

1.-30. (canceled)
 31. A method of treating a subject having a urothelial carcinoma, comprising administering to the subject an effective amount of an agent that targets and/or inhibits HER2, thereby treating the urothelial carcinoma.
 32. The method of claim 31, wherein the urothelial carcinoma is a micropapillary urothelial carcinoma (MPUC).
 33. The method of claim 31, further comprising identifying the subject, or a cancer or tumor sample from the subject, as having one or both of: (a) the presence or absence of an alteration in HER2; or (b) the presence or absence of a micropapillary histology.
 34. A method of treating a subject having a carcinoma, comprising: acquiring knowledge of: (a) the presence of an alteration in HER2; and (b) the presence of a micropapillary histology in the subject, or a cancer or tumor sample from the subject; and administering to the subject an effective amount of an agent that targets and/or inhibits HER2, wherein the carcinoma is chosen from a cancer of the urinary tract, bladder, or urothelial cells, thereby treating the carcinoma.
 35. The method of claim 34, wherein the carcinoma does not have, or is identified as not having, a gene amplification or overexpression of HER2 or a HER2 gene product.
 36. The method of claim 34, wherein the carcinoma comprises, or is identified as having, an alteration in HER2 that results in an increased activity of a HER2 gene product, compared to a wild type activity of HER2.
 37. The method of claim 34, wherein the alteration in HER2 is chosen from: (i) a substitution, a deletion or an insertion; (ii) an alteration in the extracellular domain of HER2; (iii) an alteration in domain II of HER2; (iv) a missense mutation; (v) a substitution at position 310 of HER2; (vi) a substitution of a serine residue at position 310 of HER2 to phenylalanine or tyrosine; (vii) a substitution at position 157 of HER2; or (viii) a substitution of an arginine residue at position 157 of HER2 to tryptophan.
 38. The method of claim 34, wherein the subject does not have, or is identified as not having, an elevated level of a HER2 gene product; or is negative for, or is identified as being negative for, a HER2 gene product.
 39. The method of claim 34, wherein the subject is undergoing or has undergone treatment with a non-HER2 therapeutic agent or therapeutic modality.
 40. The method of claim 39, wherein the non-HER2 therapeutic agent or therapeutic modality comprises one or more of: methotrexate, vinblastine, doxorubicin, or cisplatin.
 41. The method of claim 34, wherein the agent inhibits a HER2 gene or gene product.
 42. The method of claim 34, wherein the agent is chosen from one or more of: a kinase inhibitor; a multi-specific kinase inhibitor; a HER2 inhibitor; an EGFR inhibitor; a pan ERBB inhibitor; a small molecule inhibitor that is selective for HER2; an antibody molecule; a monoclonal or a bispecific antibody against HER2; an antibody to HER2 conjugated to a cytotoxic agent; or a HER2 cellular immunotherapy.
 43. The method of claim 34, wherein the agent is chosen from one or more of: AV-203, AMG 888, U3-1287, APC8024, DN24-02, Neuvenge, Lapuleucel-T, MM-111, MM-121, SAR256212, MM-141, LJM716, REGN1400, MEHD7945A, RG7597, RG7116, Trastuzumab, trastuzumab emtansine (T-DM1), pertuzumab, afatinib, TAK-285, Neratinib, Dacomitinib, BMS-690514, BMS-599626, Pelitinib, CP-724714, Lapatinib, TAK-165, ARRY-380, AZD8931, or Neratinib.
 44. The method of claim 34, wherein the agent is chosen from an antisense molecule, a ribozyme, a double stranded RNA, or a triple helix molecule that hybridizes to and/or inhibits a HER2 nucleic acid.
 45. A method of determining the presence of a HER2 alteration in a urothelial and/or a micropapillary carcinoma, comprising (i), (ii) or both (i)-(ii): (i) acquiring knowledge that a nucleic acid molecule comprising the HER2 alteration is present in a tumor sample from a subject; and/or (ii) acquiring knowledge of a micropapillary histology in a tumor sample from a subject; and responsive to a determination of the presence of the HER2 alteration and/or micropapillary histology, the method further comprises one or more of: (1) stratifying a patient population; (2) identifying or selecting the subject as likely or unlikely to respond to a HER2 inhibitor treatment; (3) selecting a treatment option comprising an agent that targets and/or inhibits HER2; (4) administering an agent that targets and/or inhibits HER2; or (5) evaluating the likelihood of increased or decreased patient survival.
 46. A method for screening for an agent that inhibits the expression or activity of HER2 having an alteration, comprising: optionally, determining if the alteration is present; contacting a polypeptide or protein comprising the alteration or a host cell expressing the alteration with a candidate agent; and detecting a change in a parameter associated with the alteration, wherein said parameter is selected from one or more of: (i) direct binding of the candidate agent to the polypeptide or protein comprising the alteration; (ii) a change in kinase activity; (iii) a change in an activity of a cell containing the alteration; (iv) a change in tumor present in an animal subject; or (v) a change in the level of the polypeptide or protein comprising the alteration or a nucleic acid molecule comprising the alteration.
 47. A kit comprising an agent that targets and/or inhibits HER2, or a composition comprising an agent that targets and/or inhibits HER2, with instructions for use in treating a urothelial and/or a micropapillary urothelial carcinoma, and/or instructions for determining the presence of a HER2 alteration and/or a MPUC histology, wherein the HER2 alteration is chosen from: (i) a substitution, a deletion or an insertion; (ii) an alteration in the extracellular domain of HER2; (iii) an alteration in domain II of HER2; (iv) a missense mutation; (v) a substitution at position 310 of HER2; (vi) a substitution of a serine residue at position 310 of HER2 to phenylalanine or tyrosine; (vii) a substitution at position 157 of HER2; or (viii) a substitution of an arginine residue at position 157 of HER2 to tryptophan.
 48. A purified or an isolated preparation of a nucleic acid derived from a urothelial and/or a micropapillary urothelial carcinoma, containing an interrogation position useful for determining if a HER2 alteration is present, disposed in a sequencing device, or a sample holder for use in such a device, wherein the HER2 alteration is chosen from: (i) a substitution, a deletion or an insertion; (ii) an alteration in the extracellular domain of HER2; (iii) an alteration in domain II of HER2; (iv) a missense mutation; (v) a substitution at position 310 of HER2; (vi) a substitution of a serine residue at position 310 of HER2 to phenylalanine or tyrosine; (vii) a substitution at position 157 of HER2; or (viii) a substitution of an arginine residue at position 157 of HER2 to tryptophan.
 49. A reaction mixture, comprising: a detection reagent, or a purified or isolated preparation thereof; and a target nucleic acid derived from a urothelial and/or a micropapillary urothelial carcinoma cell, which comprises a sequence having an interrogation position for a HER2 alteration, wherein the HER2 alteration is chosen from: (i) a substitution, a deletion or an insertion; (ii) an alteration in the extracellular domain of HER2; (iii) an alteration in domain II of HER2; (iv) a missense mutation; (v) a substitution at position 310 of HER2; (vi) a substitution of a serine residue at position 310 of HER2 to phenylalanine or tyrosine; (vii) a substitution at position 157 of HER2; or (viii) a substitution of an arginine residue at position 157 of HER2 to tryptophan.
 50. A method of making a reaction mixture of claim 49, comprising: combining a detection reagent, or a purified or isolated preparation thereof, with a target nucleic acid derived from a urothelial and/or a micropapillary urothelial carcinoma, which comprises a sequence having an interrogation position for a HER2 alteration, wherein the HER2 alteration is chosen from: (i) a substitution, a deletion or an insertion; (ii) an alteration in the extracellular domain of HER2; (iii) an alteration in domain II of HER2; (iv) a missense mutation; (v) a substitution at position 310 of HER2; (vi) a substitution of a serine residue at position 310 of HER2 to phenylalanine or tyrosine; (vii) a substitution at position 157 of HER2; or (viii) a substitution of an arginine residue at position 157 of HER2 to tryptophan. 